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THE BRITISH JOURNAL OF

CANCER

(The official journal of the British Empire Cancer Campaign)

THE BRITISH JOURNAL OF

CANCER

(The official journal of the British Empire Cancer Campaign)

EDITOR
R. W. ScarlT

ASSISTANT EDITOR
A. C. Thackray

Sir Samuel P. Bedson
Sir Stanford Cade
Sir Charles Dodds
Malcolm Donaldson
C. E. Dukes

B. W

ADVISORY EDITORS

Windeyer

L. H. Gray
A. Haddow
W. D. Newcomb
Sir Heneagc Ogilvie
Percy Stocks

VOL. FOURTEEN
1960

LONDON

H. K. LEWIS & CO. LTD.
136 GOWER street, W.C.l

Made and printed in Great Britain for
H. K. Lewis cfc Co. Ltd., by
Adlard cO Son, Ltd., Bartholonmc Press, Dorking.

CONTENTS

No. 1.— JIARCH

I’Ara;

yiuiR, C. S. — Cancer of the Lung. Trachea and Jvurynx in .Singapore .

Stocks, P. akd Davies. R. I. — Epiflcniiologicnl Rviflenec from Chemical and
Spectrographic Analy.sc.s- that Soil is Concenu'd in Die Can.'^afion of Cancer

Hanbuey, W. Secondary Tinnonr.s of the Heart

Englakdeb, 0 AND Sabanoi, a. — E ffect of Tliio-TEI'A on Advanced .Malignant
Ov’arian Tumours •

Dale, R. A.— The Isolation of Epithelial Cells from Normal and Neojilastie Colon

Griesbach, W. E. and Pl-rves, H. D.— RasojdiiJ Aflenomata in the Rat Hypo-
physis After Gonadectomy

Pbessov, Jil. A. — Histological and Histochemical Changes Induced by Alkylating
Agents in Transplanted RatSarcoma with Special Reference to Sarcolysine

Porter, K. A. — Graft-versus-host Reactions in the Rabbit ....

•ir,

f)i)

()(i

Caer, J. G. — Kidney Carcinomas of the Fowl Induced by the MH2 Reticnloendo-

thelioma Virus 77

Miller, J. F. A. P. — Studies on Mou.se Leukaemia. lAmkacmogcne.sis by Cell-
free Filtrates Inoculated in Newborn and Adult Mice ....

Miller, J. F. A. P. — Studies on Mouse Leukaemia. The Role of the Thymus in
Leukaemogenesis by Cell-free Leukaemic Filtrates . . * .

Powell, A. K. — .Studies on Fluid Media for the Cultivation of Mouse Ascites
Tumour Cells In Vitro .........

Lews, F. J. W. and Plaice, Constance, H. .J.— Urinary ^-Glucuronidase
Activity in Cancer of the Bladder and other Diseases ‘ .

O’Gorman, P. and Mikulska, Z. B. — ^The Antigenic Structure of a Transplanted
Sarcoma

Seelig, IL j. and Reve.sz, L. — Effectof Lethally Damaged Tumour Cells upon the
Growth of Admixed Viable Cells in Diffusion Chambers

Hidvegi, E. J., Antoni, F, and Labis, K. — Effect of Chemotherapeutics on the
N ucleic Acid Metabolism of Tumours. Incorporation of into Nucleic
Acids Following Treatment with Degranol

Kirby, K. S.— Induction of Tumours by Tannin Extracts

Survey of Papers

!)3

fl!)

lOG

121

126

139

147

CONTENTS

No. 2.— JUNE ^

Boddington, M. M., Gowdell, E. H. and Spbiggs, A. I.— Development of
Carcinoma of the Cervix Uteri. Observations Eesulting from Cvtolosical

Examination of 10,000 Cervical Smears ®

HIartins, a. G. — ^Adenocarcinoma of the Uterus in Infancy
Holland, E. _H., Acevedo, A. E. and Claek, D. A.— The Arsenic’ Content of
Bronchial Mucosa and Submucosa in Man. A Comparison of Specimens
from Lung Cancer Victims and Control Tissue .....

Baserga, E., Eutong, P. B., Tyler, S. and M^ARTiMAN, IV. B. — The Dose-response
Eelationship Between the Number of Embolic Tumor Cells and the
Incidence of Blood-borne Metastases .......

He\\gtt, H. B. .^d Wilson, C. W. — ^Further Studies Eclating to the Implications
of Eadiation Survival Curve Data for Treatment of CBA Mouse Leukaemia

by '\niole-bod 5 >- Irradiation

Bielschowsky, E. and Bielschowsky, Marianne. — C arcinogenesis in the
Pituitary Dwarf Mouse. The Eesponse to 2-Aminofluorene
Pearson, A. E. G. — Serial Irradiation of Mouse Tumours ; Some Changes in
Histologj’’ and Cj^ology .........

Israel, M. S. and Ellis, I, Eosesiary. — ^T hc Neoplastic Potentialities of Mouse
Th 5 'roid under Extreme Stimulation .......

Ghadially, F, N. — Carcinogenesis in the Skin of the Hedgehog.

Louis, C. J. — Fluorescein-globulin Affinities of the Shope Papilloma .

Belcher, E. H. and Sijipson, Shtrley M. — ^Eadioaetive Tracer Studies of Bed
Cell Destruction in Eats bearing a Transplantable Tumour
Miller, J. F. A. P. — Studies on Jlouse Leukaemia. The Fate of Thymus Homo-
grafts in Immunologically Tolerant JBce ......

Mody’, Jer K. — ^The Action of Four Carcinogenic Hj'droearbons on the Ovaries
of IF Jlice and the Histogenesis of Induced Tumours ....

PuLLiNGER, B. D. AND IvERSEN, S. — Mammary Tumour Incidence in Eelation to

Age and Number of Litters in CaH/ and EIII/ Mice

Pdllinger, B. D.' — ^Tests for Mammarj' Tumotir Agent in C 3 H/ and EIH/ Mouse
Strains

Young, Stretton and Fraser, Lucy B. — Experiences with Hj^iophysectomy in
Jlice. Criteria of Complete Eemoval .......

Daaues, W. and Wiljishubst, j. E. — Carcinogens formed in the Heating of Food-
stuffs. Formation of 3,4-Benzopyrene from Starch at 370-390° G. . .

Stein- Webblowski% Eachel. — I nduction of Cancer of the Cervix Uteri in
Eelation to the Oestrus Cycle

Drayton, H. A. — The Effect of Incubation on Three Fowl Tumour Viruses
Allison, A. C. and Armstrong J. A. — ^Abnormal Distribution of Nucleic Acid in
Tissue Culture Cells Infected with Polyoma Virus . . . • •

Montesiurro, D. G. — The Effect of Nitrofurazone on the Testes and Accessory
Sex Organs of Normal Eats and Eats bearing the Walker Garemoma 256
Clark, Catherine M. and Goodlad, G. A. J. — ^The Influence of Diet on the
Action of the Wallier 256 Carcinoma on Liver Protein and Nucleic Acids .
O’GoRaiAN, Patrick. — ^T he In Vitro Cj'^totoxic Activity of Murine Iso-antisera
on Normal and Malignant Cells . . • • _ • ' . L '

MtRVTSH, S. AND GiLLMAN, J. — Bile Acid Composition and Bile Volume in Butter
Yellow Fed Eats in Eelation to Liver Cancer . . . . •

CsABA, G., Horvath, Cecilia and Acs, Th. — S ome New Data concerning the
Biology of Tumours. The Effects of Heparin and its Components on

Tumour Growth

CsABA, G., Acs, Th., Horvath, Cecilia and Napa, Eszter.— S ome New Data
concerning the Biology of Tumours. The Effects of Heparin Inhibitors on
Tumour Growth . . . . • • • • • ‘

Lemon, H. M., Mueller, J. H., Looney, J. M., Chasen, W. H. and

Marcia. — H j'percitricemia in Human Cancer. Factors concerned m Patno-
genesis and Treatment

Survey of Papers . .

CONTENTS

^lo. 3 .— SEPTEMBER

PAGK

Stocks Perct. On the Relations between Atmospheric Pollution in Urban and
Rural Localities and Mortality from Cancer, Bronchitis and Pneumonia,
with Particular Reference to 3 : 4 Benzopyrene, Beryllium, Molybdenum,
Vanadium and Arsenic ..•••••••

Staszewski, J.— Smoking and Cancer in Poland . . . • ■ •

DE WaAED, F., DE LaI\T3, J. W. j. and BiVANDERS-VAN HALEWIJN, E. A.

On the Bimodal Age Distribution of Mammary Carcinoma
Herdan, G. — ^The Frequencj' of Cancer in Diabetes Mellitus . . . •

Patey, D. H.—Early (Prophylactic) Oophorectomy and Adrenalectomy in

Carcinoma of the Breast ; An Interim Report

Sim, a. W., Hobkirk, R., Stewart, Heeen J., Blair, D. W. and Forrest,
A. P. M. — ^Urinary 17-Ketosteroids and their Fractions in Women uith

Breast Cancer Treated by Endocrine Surgery

Whitaker, B. L. — Plasma ^-Glucuronidase Levels in Breast Cancer

Heath, J. C.— The Histogenesis of Malignant Tumours Induced by Cobalt in the

Rat

IiiJiAN, 0. AND Ghadully, F. N. — Coat Colour and Experimental Melanotic
Tumour Production in the Hamster .......

Cherry, Cora P. and Glhcksmann, A. — ^The Effect of Endocrine Changes, of
Irradiation and of Additional Treatment of the Sldn on the Induction of
Tumours in the Female Genital Tract of Rats bj^ Chemical Carcinogens .
Pullinger, B. D. — ^Reduction of Mammary Carcinoma and Adenoma in C^Hf
Breeders after Late Ovariectomy .......

Ranadive, Kajlal j., Hakdi, Saeta A. and Kharkar, Kumud R. — Chemical
Induction of Mammarj' Cancer in Pseudopregnancy ....

Maechant, June. — ^T he Development of Ovarian Tumours in Ovaries Grafted
from Mice Pretreated with Dimethylbenzanthracene. Inhibition by the
Presence of Normal Ovarian Tissue .......

Maechant, June. — ^T he Development of Ovarian Tumours in Ovaries Grafted
from JiEce Pretreated with Dimethylbenzanthracene. The Effects of the
Grafting Operation Itself and the Effects of Grafting Ovaries from Mice at
Earlj' Stages in Pretreatment vith the Carcinogen .....

Darcy, D. A . — A Quantitative Study of a Serum Protein Associated ivith Tissue
Growth. Levels found in Rats under Various Physiological Conditions .
Darcy, D. A. — ^A Quantitative Study of a Serum Protein Associated with Tissue
Grondh. Values found in Tumour-bearing Rats .....

Sahasrabudhe, M. B., Neburkar, M. K., Naburbar, M. V., Tilak, B. D. and
Bhavsae, 31. D.' — Inliibition of Tumour Groirth by Interference of Hexose-
mono-phosphate Pathway. Synthesis and Anticancer Properties of
Thiophene 2 : 5 Dicarboxyiic Acid •....,.

Thomlinson, B. H. — ^An Experimental Method for Comparing Treatments of
Intact 3Iah‘gnant Tumours in Animals and its Application to the Use of
Oxygen in Radiotherapy .........

Ball, J. K. and 3IcCaeter, J . A . — A Study of Dose and Effect in Initiation of Skin
Tumours by a Carcinogenic Hydrocarbon ......

Huh, Tai-Young and 3IcCarter, J. A.— Phenanthrene as an Anti-initiatine

Agent . ®

SuEimv OF Papers ...

SQ7

419

437

449

457

4G0

471

478

483

489

502

508

514

519

524

634

547

555

577

591

CONTENTS

No. 4.— DECEMBER

Muib, C. S. and Kirk, R. — Betel, Tobacco, and Cancer of the Mouth

Ferrari, E. and ICreyberg, L. — Histological Tj7)es in a Lung Cancer Material
in ^^enice ...........

Thackeav, a. C. and Lucas, R. B. — The Histologj^ of Cylindroma of Mucous
Gland Origin

Brew, D. StJ. and Jackson, J. G.— Lj^mphosarcoma in the Ovary in Young
African Girls in Nigeria .........

Greening, W. P., Ramsay, G. S., Stevenson, J. J., Boyland, E., Rigby-Jones,
P. C. AND Godsmark, B.— Results in the Treatment of Cancer of the
Breast by Interstitial Irradiation of the Pituitary'' .....

Hirchinson, Vivienne and Hide, K. R.— The Effects of a Seneeio Alkaloid
(Monocrotaline) on Human Embryo Liver in Tissue Culture .

Ghadially, F. N., Illjian, 0 . and Barker, J. F. — The Effect of Trauma on the
Melanotic Tumours of the Hamster .......

Clemo, G. R. and JIilder, E. W. — Tumour Promotion b3’' the Neutral Fraction
of Cigarette Smoke ..........

Howell, J. S. — The Chemical Induction of Breast Tiunours in the Rat : Hor-
monal Factors in Tiunour Production .......

Biancitiori, C. and Caschera, F. — The Gradual Conversion of a Spontaneous
Mouse IMammarj' Carcinoma to an Ascites Tumour ....

Negroni, G. and Chesterman, F. C. — Virus-cell Relationship in Kidney
Tumours Induced in Golden Hamsters bj’’ the Mill Hill Potyoma Virus .

Stoker, JIichael. — S tudies on the Oncogenic Activitj' of the Toronto Strain of
Poljmma ^^irus ...........

Bitensky, Lucille, Baldwtn, R. W. and Chayen, J. — A Histocheniical Study
of the Earlj'^ Stages of Carcinogenesis in Rat Liver : Localization of Fluores-
cent Carcinogen and Changes in Succinic Dehj'drogenase Activitj' .

Bitensky, Lucille, Baldvtn, R. W. and Chayen, J. — A Histochemical Study
of the Earlj'^ Stages of Carcinogenesis in Rat Liver ; Changes in Cellular
Lipids and jlitochondria .........

Lyons, M. J. and Spence, J. B. — Enviromnental Free Radicals

Houghton, P. and Martin, N. H. — The Mj^eloma Globulins . . . .

Roberts, D. C. and Trevan, D. J. — Cell Division in an Ascites Tumour in vitro
with Especial Reference to Abnormalities of Cytoplasmic Cleavage .

Trevan, D. J. and Roberts, D. C. — Sheet Formation bj'^ Cells of an Ascites

'' Tumour iv vit)‘o . . . . . . . . . •

Siegel, E., Sachs. B. A. and Graig, F. A. — Fate of ^^S-Cj'^stine in Multiple
Mj^eloma . . . . . . . . • ■ ■ . ■

Neish, W. j. P. and Rylett, Ann. — A ccimiulation of Phosphoethanolamine in
the Livers of Rats Injected with Hepatocarcinogens . . • ■

Calcutt, G., Doxey, D. and Coates, Joan. — ^T he Effects of Some Chemical
Carcinogens upon the — SH Levels of Target and Non-target Tissues .

Butler, J. A. V. and Laurence, D. J. R. — ^Relative Metabolic Activities of
Histones in Tumours and Liver

Index

Survey of Papers

PAGE

597

609

612

.621

627

637

647

651

657

668

672

679

690

696

703

709

716

724

730

737

746

758

764

BRITISH JOURNAL OF CANCER

YOL. XIY MAl^Cll, 1960 NO. 1

CANCER OF THE LUNG, TIUCHEA aVND LARYNX IN SINGAPORE

0. S. MUIR

From the Department of Patholoc/i/, University of Malaya in Singapore
Hceoived for jiiiblicntion Doceinbor 18, Ifl.lO

Caxcer of the lung is licld to be increasing in incidence, probablj'’ on a world-
ivide basis. Tliis paper presents data on cancers of the lung, trachea and larynx
based on the post-mortem protocols of the University and Government Depart-
ments of Pathology. Singapore, and on the reports of the Registrar-General,
Singapore, in the hope that the information adduced ivill prove useful as a base-
line for subsequent eindemiological cancer re.search in this ai'ea.

AIETIIODS AND MATERIALS

The post-mortem records of all cases of cancer of the lung, trachea and larynx
for the years H)4S-.'58 inclusive were individually scrutinised and the relevant
data entered on a pro-forma for subsequent analysis. Nearly all post-mortems
in Singapore are performed bj' the staffs of the two departments of Pathology.
These embrace both hospital deaths and deaths coming within the aegis of H.M.
Coroner, Singapore, i.e. all violent, sudden and unnatural demise.

The age and sex distribution of both the Singapore and the post-portem
population for the period under review have been published previously, and the
probable bias and error inherent in the material indicated (Muir, 1959). Briefly,
although there is a multiracial population of Malays, Chinese. Indians and Others
the post-mortem rates for these races are not proportipifpil to their absolute
numbers, due to religious and other factors, and further, there is some aversion,
particularly among the Malays, who tend to live in rural Singajjore, to seek medical
advice. It is probably true to say that this latter factor will decrease in
importance over the next decade.

The lung

There were 173 cases of primary cancer of the lung. Tliis represents 0-75
per cent of the 22,997 post-mortems carried out in 1948-58, or if the 11,525
children under the age of 11 years be excluded, 1-5 per cent.

There were 139 males. The mean age at death was 53-4 ± 10-7 years. (One
boy aged 11 years, and 2 males of unknown age are not included in this figure.)
vli these 129 were Chinese, 5 were Europeans (mean age 55 years), 3 were Tamil
ndians (mean age 43 years) and 2 were Malay (mean age 37 years).

Of the 34 females, whose average age at death was 53-8 ± 13-7 years, 31 were

nnese. There were also one Eurasian, one Japanese and one Tamil Indian.

C. S. MUIR

The male/female ratio is thus 4 to 1. The age distribution by sex for the
Uiinese is shoyTi in Pig. 1, and is compared with that of a series of 1749 lunfr
cancers seen at the Brompton Hospital, London (Bignall, 1968). ®

It will be seen that although the maximum incidence in London and Singapore
IS in the decade 50-59, there are relatively more deaths before the age of 40 and
between 40-49 in Singapore.

The primary tumour was in the left lung in 64 (37 per cent), in the right in
104 (60 per cent), and at the tracheal bifurcation in 2 (1 per cent). In a further

Pig, 1.- — Comparison of the age distribution, by sex, of the Singapore Chinese with lung cancer,
with a series from the Brompton Hospital, London (Bignall, 1958).

X X Chinese males. • • English males.

X X Chinese females. • • English females.

2(1 per cent) it was impossible to state in which lung the primary tumour arose.
There was one case of pleuro-endothelioma, in the boy aged 11 years mentioned
above.

In the left lung 26 per cent of tumours arose from the main bronchus, 48 per
cent were found in the upper lobe, 20 per cent in the lower lobe. In the remaining
6 per cent the site was not, ,or could not, be specified. In the right lung 30 per
cent arose from the main bronchus, 30 per cent were found in the right upper lobe,
5 per cent in the middle lobe and 28 per cent in the lower lobe. In 7 per cent the
site was not specified. These figures are in general accord vdth the observations
of Bryson and Spencer (1951).

Intrapulmonary metastases were very common, and in 20 per cent involved
the opposite lung. Proportionately, neither lung seemed to have spread to its
fellow more often than the other. Ochsner and de Bakey (1942) recorded
metastasis in the opposite lung in 23 per cent.

LUNG CANCER IN SINGAPORE

The ratio betAveen tiiiuours of the right and left lungs is as in most occidental
reports (Ochsner and de Bakey, 1942).

jMetastases were common. The mediastinal glands were grossly involved m
66 per cent, the cervical and supraclavicular glands in 16 per cent. Spread to
the upper abdomhial nodes was recorded in 12 per eent. These figures are some-
what lower than those of Ochsner, Dixon and de Bakey (1945).

Pericardial metastases were present in 20 per cent ; haemopericardium,
often exceeding 200 ml., in a further 6 per cent ; cardiac secondaries were seen in
2 per cent. The superior vena cava was invaded in 10 per cent and significantly
compressed in a further 10 per cent. The pulmonary artery, the aorta, and other
great vessels were invaded in 3 per cent. One case of direct fistula between
aorta and bronchus was recorded. Bony spread (15 per cent) was most often
seen in the ribs, the femur and the frontal bones of the skull. Femoral fracture
occurred in 6 per cent.

Pancreatic, thyroid, splenic and peritoneal spread occurred in 3 per cent.

Onuigbo (1957) stud3mig 1000 lung cancer autopsies found suprarenal meta-
stases in 38-5 per cent, hepatic in 42-6 per cent and Iddney in 17-3 per cent, the
adrenal and liver figures being 10 per cent higher than those of tliis series.

Discussing the mode of spread of the neoplasm he found that 61 per cent of
single adrenal secondarj'- tumonrs were on the same side of the body as the primary
tumour, 39 per cent on the other, concluding that spread to the adrenals must be
lymphatic. Comparable figures for tliis series are 79 per cent and 21 per cent.
This difference, although based on smaller numbers, is also significant. Many
more of the adrenal metastases were bilateral, in all 60 per cent. In several the
ipsilateral was noted to be larger in size than the contralateral, but more often
than not no mention was made as to the relative dimensions.

Onuigbo (1957) found that 65 per cent of renal metastases were ipsilateral.
In this material renal metastases were seen in 17 per cent, 37 per cent of winch
were ipsilateral, 37 per cent contralateral and the remaining 26 per cent bilateral.
Brain spread was noted in 24 per cent, being ipsilateral in 39 per cent, contra-
lateral in 32 per cent and on both sides in 29 per cent. Meyer and Reah (1953)
found cerebral metastases in 25 per cent of their necropsy cases.

The figures for extrathoracic metastasis in this series are in close agreement
■^vith those of Bryson and Spencer (1951). Pleural effusion was seen in 25 per
cent, two-thirds of which were blood-stained.

Trachea

There were two cases of neoplasm of the trachea. One in a 16-year old
Chinese boy, which straddled the trachea at the level of the suprasternal notch,
infiltrating surrounding muscle and the oesophagus, proved to be a round cell
sarcoma. The other arose 5 cm below the vocal cords in a Chinese male aged
41 years, infiltrating the outer oesophagus. Histologically the tumour was of
epidermoid origin. Tracheal tumours are rare ; they have been well described
by Culp (1938).

Larynx

There were 12 cases of laryngeal carcinoma, one in a Tamil Indian woman
aged 47 j^ears ; the remamder in Chinese males, whose mean age at death was
o2-o ± 6-7 3'^ears.

C. S. MUIE

17 per cent of t]ie tumours u^ere found on the left vocal cord, 17 per cent on
the right. 25 per cent involved both cords and 25 per cent arose from the
ivhUe 1 1 per cent involved both the vocal cords and the epiglottis
Lymph node involvement was noted in 50 per cent of cases, and was usually
bilateral. Spread to the lung was seen in 25 per cent, to the liver in 17 per cent.
The incidence of metastasis is high, spread from tumours of the endolarjmx
usually bemg rare (Ackerman and Eegato, 1954). This is no doubt due to the
extensive nature of most of the tumours.

In the five-year period 1954-58 neoplasms of the larjmx accounted for 2-0
per cent of hospital admissions ndth malignant disease, 2-2 per cent of hospital
cancer deaths, 0-6 per cent of post-mortems on cases of malignant disease, and
l‘S per cent of cancer deaths recorded by the Registrar-General (Singapore).

In 1940-42 1-7 per cent of all cancers recorded by the Registrar-General,
England and Wales, were in the larynx (Kennaway, 1950), the ratio of larynx to
lung cancers being 1 ; 4-4. In Singapore for 1954-58 mclusive the ratio is 1 : 6-2.

These tumours are thus substantiallj’- as seen elsewhere and ndll not be
discussed further.

DISCUSSION

Hoffman (1915) quoted, from the Annual Reports of the Medical Department
of the Straits Settlements, figures of relative incidence for the 121 cancer cases
seen at Tan Took Seng’s Hospital, Singapore, from 1907 to 1912 inclusive. 5-8
per cent of these were in the lung. If we can accept that a further 4-1 per cent
described as “ sarcoma of the mediastinum ” may have been oat cell or undifferen-
tiated carcinomata, the oA'erall incidence was then 9-9 per cent.

The late Dr. J. C. Tull, sometime Senior Pathologist, Singapore, provided
comparative data for a paper by Bonne (1937) on the incidence of malignant
disease in South-East Asia. He found that 6-2 per cent of malignant tumours
at post-mortem were in the lung. In 1948-58 inclushm, in a total of 1096
malignant tumours seen at autopsy, 176 or 16-7 per cent arose in the lung.

Prior to 1954 tumours of the larynx, lung and trachea were not separated
in the returns of both the hospitals and the Registrar-General (Singapore).
Since then cancers of the lar3mx (List Ho. 161) have been entered separately, but
trachea is still included with lung (List No. 162-3). It is felt that the number of
tracheal tumours is negligible and henceforth in this paper when the word lung
is used in coimection with hospital and Registrar’s figures, strictly it should read
lung and trachea.

The crude death rate for cancer of the lung for 1954^58 was 0-066 per 1000
living per annum. A comparable rate for England and Wales for 1951-55 was
0-355 per 1000 living per aimum. As the Singapore population has such an
unusual structure, more than half being under the age of 21 years, and as t le
age specific death rates are not at present available for individual tumours,
although available by sex for neoplasms as a whole, further comparison seems

In the quinquennium 1954—58 there were 7131 admissions to hospital Avith
some form of malignant disease (List No. 140-205). Of these tumours 10-8 per
cent were diagnosed as being in the lung. 21-4 per cent of those admitted died,
and of these deaths 16-4 per cent were due to lung cancers. In tins period 660

LUNG CiUSTCER IN SINGAPORE

post-mortems were carried out on cases of malignant disease, 15-2 per cent of
which were found to be pulmonarj'^ in origin. Tire Registrar-General (Singapore)
noted that 10-9 per cent of all deaths due to malignancj^ were notified as lung
cancers. Cancer of the lung would thus appear to account for between 10 and
15 per cent of all malignant tumours in Singapore. Kennaway (1950) gives an
abstract of deaths from malignant disease by form and site, in England and Wales
for 1940-42, based on the retm-ns of the Registrar-General : cancer of the lung
accounted for 7-6 per cent of carcinomata.

City life is held to increase lung cancer morbidity (Stocks, 1959) and although
a large proportion of the Singapore population lives in the city proper, the
atmosphere is but little polluted by heavy industry which is, as yet, virtually
non-existent. There is, however, a high density of vehicular traffic propelled by
petrol and diesel engines, manj^ of wliich emit excessive exhaust.

In Britain, for the period 1951-55, Doll (1958) gives the mean annual consump-
tion of cigarette tobacco for males over the age of 14 years as 7-8 lb. (3-5 kg) ;
for females as 2-6 lb (1-2 kg). The mean value for both sexes, assuming sex
parit3’-, being 5-2 lb. per person (2-4 kg). In Singapore, a eomparable mean value
for both sexes over the age of 14 j’^ears, based on issues from bond for consumption
on the Island of both imported and locally manufactured cigarettes, for the years
1956-58 inclusive, is 6-2 lb. (2-8 kg) per person per annum. As many children
under the age of 15 j^ears appear to smoke perhaps the mean figure of 3’7 lb.
(1-7 kg) of cigarette tobacco per person per annum may be of more significance.
The consumption of pipe tobacco is low, 0-12 lb. (0-06 kg) per head of population.

An unknown number of persons in Singapore smoke opium, a drug to which
occidentals are not usuallj’’ exposed. Of these 633, all males, were sent in 1957
to the Opium Treatment Centre for rehabilitation. Whether opium has any
carcinogenic effect is, of course, another matter.

Cancer of the lung is not unknomi in the other countries of South-East Asia.
Vellios, Goonchom and Suvanatem^a (1953) described the tumours seen in two
hospitals in Thailand. In a ten-month period 198 autopsies were performed, in
which material there was but one lung cancer, and tliis an incidental discovery.
This was the only lung tumour seen in 350 malignant neoplasms, both post-
mortem and biopsy.

Piyaratn (1959) reviewing 1100 biopsies of malignant tumours (and including
some of those already described by Vellios et al. (1953)) submitted to the
Department of Pathology, Chulalongkom Hospital, Bangkok, found 24 lung
tumours, 20 of which were in males. Erom this evidence he states “ ... that

bronchogenic carcinoma is rather common in Thailand ... ”, but does not

attempt to relate his material to the Bangkok population as a considerable number
of patients came from without the city.

In Ceylon, Cooray and Leslie (1958) found 5 bronchial carcinomas in the
2562 post-mortems carried out in the five years 1952-56 — an incidence of 0-2
per cent. A fruther 22 cases were diagnosed on biopsy. The post-mortem
incidence is thus somewhat lower than in Singapore, as is the consumption of
tobacco.

Kouwenaar (1951) describes a post-mortem series of 1301 Chinese and 1189
Javanese males in Sumatra. Malignant disease was found in 120 and 59 respec-
tivelJ^ Lung cancer accormted for 9 per cent of the malignancies in the Chinese
and for just under 1 per cent in the Javanese. Marsden (1958) in his survey of

c. s. :muir

4650 biopsy malignant neoplasms seen in Malaj^a, finds that 7-8 per cent of
mahgnant tumours in the Chinese male, and 2-1 per cent in the Chinese female
are pulmonarj’-. Figures for the Malay and Indian of both sexes, are respective^
2-9 and l-l per cent, 3-8 and 1‘3 per cent. These observations are of some import,
as from the figures of the author and those from Sumatra it might be assumed that
lung cancer is rare in the Malajn

Sliih e( al. (1959) discuss, from the clinical aspect, 236 cases of bronchogenic
carcinoma seen in Shanghai from 1949—57 inclusive. These cases were proven b}'^
biopsy or cjdology ; a further 700 diagnosed by X-raj^ alone were not described
further. The sex ratio was 3-5 ; 1, males predominating. 16 per cent of patients
were below the age of 40 ; considerably more than the Brompton Hospital series
and slightlj'^ more than in Singapore. 55 per cent of these patients were noted
to be habitual smokers.

Yell and Cowdry (1954) examined 1869 malignant tumours collected in
Taiwan (Formosa). These comprised 1729 biopsies and 140 autopsies on persons
with malignant disease. A total of 19 lung cancers were encountered, 3 post-
mortem, 16 removed surgically, or 1-02 per cent of all the tumours. The peak
incidence of these tumours was, as in this series, in the decade 50-59. The sources
of bias in tliis material are full}’’ discussed.

Stransky and Felix (1951) found 1 lung cancer in 61 necropsies on persons
with malignant disease performed between 1911 and 1919 at the Philippine
General Hospital, Manila. Between 1945 and 1950, at the same hospital, there
were 141 cases of malignancy with post-mortem examination, lung tumours
comprising 4 per cent. In a total of 919 malignant tumours diagnosed on biopsy
at the University of San Tomas by Sta. Cruz and de Jos Santos (1955) between
1946-53, 0-3 per cent were in the lung. In the 122 cases of cancer who came to
post-mortem, S'4 per cent of all autopsies, 4 (3 per cent) were pulmonary in
origin.

In brief, cancer of the lung is found tlnoughout Asia. At present its incidence
at large seems to be low, although where figures are available about one-tenth of
all deaths fi’om mahgnant neoplasm are in the lung, a value not far removed from
the reported relative incidence in the West.

SmUMAKY

The main morbid anatomical features of the 176 lung, the 2 tracheal, and the
13 laryngeal cancers seen in the 22,997 post-mortems performed by the University
and Government Departments of Pathology, Singapore, from 1948-58 inclusive,
are described, and are seen to be as elsewhere.

About one-tenth of all cancers admitted to hospital, and of all cancers regis-
tered by the Registrar-General (Singapore) from 1954-58 inclusive were m le

In 15 per cent of all post-mortems on persons with mahgnant disease, the

primary tumour was of pulmonary origin. ro • i •

The crude death rate for cancer of the lung in Singapore for 1954-58 me usive

was 0-065 per 1000 living per aimum. .

More cases of lung cancer are seen before the age of 40 than •

The consumption of tobacco in Singapore is noted to be 3-7 lb. ( g) per

person per annum.

LUKCt cancer in SINGAPORE

A portion of the literature on cancer of tlie lung in South-East Asia and in
China is discussed.

I nnsh to thank Professor R. Kirk for land help and encouragement, my
colleagues of the Government and University Departments of Pathology for
access to their post-mortem notes and records, IMr. E. J. Phillips and IMr. S. C.
Chua of the Department of Statistics, Singapore, and Mr. Lee of the Customs
Department, Singapore, for various data, IMr. Ti Teow See for Fig. 1, and Jlr. P. A.
Samuel who tj^Ded the script. This communication forms part of a thesis for the
degree of Ph.D. (Malaya).

REFERENCES

Ackeejias, L. V. AJxB Regato, j. a. del. — (1954) ‘Cancer. Diagno.sis, Treatment and
Prognosis’. 2nd Edn. London (lOmpton), p. 416.

Bignall, j. R. — (1958) in ‘Monographs on Neoplastic Disease. Vol. 1. Carcinoma
of the Lung’. Edinburgh (Livingstone), p. 285.

Boio^e, C. — (1937) Amer. J. Cancer, 30, 435.

Beysox, C. C. aed Spencee, H. — (1951) Quart. J. Med., 20, 173.

CooEAY, G. H. ajnD Leslie, N. D. 6. — (1958) Brit. J. Cancer, 12, 1.

Culp, 0. S. — (1938) J. thorac. Surg., 7, 471.

Doll, R. — (1958) in ‘Monographs on Neoplastic Disease. Vol. I. Carcinoma of the
Limg’. Edinburgh (Livingstone), p. 71.

Hoffman, F. L. — (1915) ‘The Mortality from Cancer throughout the World’. Newark,
New Jersey (The Prudential Press), p. 712.

Kennaway', E. L. — (1950) Brit. J. Cancer, 4, 158.

Kouwenaae, W. — (1951) Docum. neerl. indones. Morb. trap., 3, 357.

Maesden, a. T. H. — (1958) Brit. J. Cancer, 12, 161.

Meyee, P. C. and Reah, T. G.— (1953) Ibid., 7, 438.

Muie, C. S.— (1959) Ibid., 13, 595.

OCHSNEE, A. AND De Bakey, M. — (1942) J. thorac. Surg., 11, 357.

Idem, Dixon, J. L. and De Bakey, M. — (1945) Clinics, 3, 1207.

Onuigbo, W. I. B. — (1957) Brit. J. Cancer, 11, 175.

PiYAEATN, P. — (1959) Cancer, 12, 693.

Shih Mei-Hsin, Ku Suei-Yueh, Chang Chiu-Ping, Ku K’Ai-Shih, Tiao Yu-Tao and
Chu Eeh-Mei.— (1959) Chin. med. J., 79, 19.

Sta. Ceuz, j. Z. and De Los Santos, R.— (1955) J. Phil. Is. med. Ass., 31 637
Stocks, P.— (1959) Practitioner, 182, 667.

Steansky, E. and Felix, N. S.— (1951) J. Phil. Is. med. Ass., 27, 106.

Vellios, F., Goonchoen, S. G. and Suvanatejuya, P. — (1953) Cancer, 6 188
Yeh, Shu and Cowdey, E. V.— (1954) Ibid., 7, 425.

EPIDEMIOLOeiCAl EWDBNCE FROM CHEfflCAE ASD SPECTEO

Soroi^SEr^ cok„t?h°e

P. STOCKS AND R. I. DAVIES

From the Department of Agricultural Chemistry, University Ocllege of North Wales,

Bangor

Received for publication December 5 , 1959

The reasons which led to the initiation in 1952 of a stud}^ of the cliemical
properties of garden soils in connection with cancer in North IVales and Chesliire
have been detailed (Stocks, 1957, 1958). TJie Cheshire and North Wales Branch
of the Campaign liad included soil amongst the environmental items to be
investigated in the 4-year cancer survey, and with their help the Department of
Agricultural Ghemistrj'’ was enabled to undertake the chemical analyses, and at a
later date spectrographic estimations of trace elements. Collection of soil Samples
from gardens of houses where a death of a resident had occurred from cancer or
other cause was made possible by the co-operation of the Medical Officers of
Health and was carried out hy their Public Health Inspectors.

Since 1957 the laboratory work on some 2,000 soils has continued with the
aid of a grant to the University College of North Wales, a special stud}’- has been
made also of two districts with liigh and low incidence of stomach cancer in
Devonslure, and analytical work on plants and spectroscopic study of polycyclic
h 3 ^drocarbons have been started. Brief progress reports have appeared in the
Annual Reports of the British Empire Cancer Campaign for 1955 to 1958, and the
purpose of this paper is to present a more complete and up-to-date account of the
outcome so far of the work on soils, including the Devonshire study.

A hj’pothesis that one of the causes of cancer of the stomach is in some way
connected with soil cannot be dismissed on the grounds that we do not know how
such a connection could arise. Kant once remarked that “ Philosophy is often
much embarrassed when she encounters certain facts which she dare not doubt
yet -will not believe for fear of ridicule The soil hypothesis has met with
scepticism from those who look for a single “ cause ” of cancer, but as Sir Julian
Huxley (1958) vTote in discussing the statistical evidence about lung cancer,

“ It is surely time that we should drop mediaeval concepts concernuig causation
and think in terms of multiple correlation ”. Having established as a first step
the existence of statistical correlations between the incidence of stomach cancer
and the amounts of organic material and certain trace elements in the neighbour-
ing soil- possible reasons for this such as vegetables, water supply, air pollution
and radioactivity have next to be investigated.

Organic Content of Soil in Districts having Different Rates of
Stomach Cancer Mortality

In the North Wales and Chesire region where soils contain inconsiderable
amounts of free calcium carbonate the loss of weight on ignition of a dry soil,
expressed as a percentage, provided an index of the organic matter present.
HTiere the soils are contaminated with coal or ashes the loss on ignition would be

SOIL AND THE CAUSATION OF CANCER

affected, .so a second index was obtained of the readil}' oxidisable or “ organic
carbon ” by a wet clieinical oxidation of the soil (Walkley and Black, 19,34).
In this method, coal and ash arc but little affected and so do not contribute to the
index. Soils seen on examination in the dried state to be appreciably contamin-
ated by small coal have, however, been dealt with separately in the statistical
analysis.

During the years whilst soil samples were being collected the aim was to obtain
a sample from every house where a death occurred from c<ancer of the stomach,
intestine, rectum, larynx, lung or breast or from leukaemia or Hodgkin’s disease,
and in the counties of Anglesey. Caernarvon and Cheshire omissions arose only
through absence of a garden or inabilitj' to contact relatives or obtain the necessary
permission. In the other counties the coverage was not so complete owing to
pressure of work on the Health Officers, but such selection of cases as resulted
was connected with periods of time and not with the nature of the cases.
Histories of all persons dying of cancer were obtained independently from rela-
tives if no hospital history bad been obtained previously, and this information
included the duration of residence at the bouse prior to death. In order to
obtain a control series addresses were taken from the monthly lists of deaths
from causes other than cancer by matching the sex, age and district of residence
■nith the first GOO stomach cancer cases as nearly as was practicable.

The soil stud}"^ covered the oo administrative areas in North Wales and 11 in
the parts of Cheshire within the Liverpool Hospital Region, excluding the county
boroughs, and details of these were given bj”^ Stocks (19.57, Table 6 and Map 2).

The standardised mortalitj’’ ratios (S.M.R.’s) for cancer of the stomach in the
8-year period 1947-55 were given in the final column of that table and depicted in
Map 3, and they ranged from 65 to 262 compared with 100 for England and Wales,
four very small districts with fewer than 5 deaths being combined Avith adjoining
areas. On page 104 of the report a general similarity between the geographical
distribution of stomach cancer mortality and organic carbon content of soils
not directlj'^ associated with cases of cancer was pointed out, Avhereas no such
correspondence was apparent Avith cancer mortality rates for intestine, lung or
breast.

When the districts are diAuded up into 9 groups Avith stomach cancer S.M.R.
•50-, 75-, 100-, 125-, 150-, 17.5-, 200-, 225-, 250-274, and soils distributed on scales
of organic carbon and ignition loss, the correlation coefficients betAveen organic
content and the mortality from stomach cancer in the district where the soil Avas
taken are as shoAAm in Table I,

Table I. — Correlations Between Measures of Organic Matter Content of Soils not
Directly Connected with a Case of Stomach Cancer and the Level of Mortality
from Stomach Cancer in the District from which the Soil was taken. Estimated
from Tioo Alternative Series of Samples

Organic carbon
Agnition loss*

(1) Non-cancer
control soils

t \

Number Mean S.D.

.S13 2-98 1-22

373 9-34 4-12

(2) Non-gastric
cancer soils

Number Mean S.D.

1063 2-86 1-35

681 9-24 4-09

Correlation with S.M.Il.
from stomach cancer in

1st Series 2nd series
0-4661 0-4015

0-5693 0-5027

included the coefficients become 0-481 in the non-caneer

ana 0-402 in the other series.

P. STOCKS AND E. I. DAVIES

Table II Organic Carbon Content of Soils from Houses with Stomach Cancer, showing Excess
over the Calculated Frequencies based on the Corresponding Distributions of Other Cancer
and H on-cancer Cases {corrected for the Relative Mortality from Stomach Cancer in the District).

Years

house
Under 10

10-19 .

20 and over

Organic
carbon
per 100
0 -
2 -
3-5-
5 +

Total

0 -
2 -
3-5-
5 +

Total

0 -

2 -

3-5-
0 +

Total

No. of soils grouped according
to S.M.R. of district

50- 100- 125- 150- 175-200+

18 18 7 3 3 2

12 ID 15 18 5 12

4 4 7 8 6 4

— 1 4 5 2 4

Sub-

totals

Excess as calculated from

I ^

Non- Other Non- Other

34 42 33 34 16 22 181 . 109 72

8
4
4

32 35 23 34 15 23 162

90 72

126

29
56

30
17

38 44 50 75 33 41 281

132 149

2-8

1-1

4-5

-9-0

4-0

0-6

13-0

-18-6

0-7

13-0

8-1

3-3

- 6-6

3- 0
-0-4

4- 7

- 8-8

4- 4
2-0
8-0

5- 6

32-7 32-9

6-1 3-1
3-5 10-6
9-2 5-5
15-3 14-1

0-7 34-0 33-3

-4-2

42-5

9-0

17-1

- 6-6

44-4

18-6

10-2

64-4 66-6

Under 10

10-19

20 and over

Organic carbon distributions of soils from houses in same
groups of districts where a death had ocurred from :

Non-cancer cause

( —

—

50-

100-

125-

150-

175- 200+

All

50-

100-

2- .

3-5- .

5+ -

—

■ —

Total .

115

2- .

3-5- .

5+ .

—

. —

—

Total .

3-5- .

5+ .

—

Total .

181

Other cancer (i.e. not stomach)
'

64 48 23 43

47 36 20 18

73 79 31 29

All

102

156

352

121

257

138

102

360

SOIL AIS’D THE CAUSATION OF CANCER

Table III .— on Ignition of Dried Soils from Houses with Sto?nach Caiicer, Showing Excess
over the Calcxdated Frequencies based on the corresponding Distributions of Other Cancer
and Non-cancer Cases {Corrected for the Relative Mortality from- Stomach Cancer in the

District)

Excess as calculated from

Years

Loss on
ignition
per 100

No. of soils grouped according
S.JI.R. of district

Sub-

totals

Non-

Other

A-

Non-

Other

house

50-

100-

125-

150-

175-

200-1-

All

50-

150-1-

' 50-

50-

150 -f-

150-f’

der 10

14 .

-6-7

0-6

0-7

5-2

S-5-

15 .

0-1

-0-6

6-6

2-8

11-5-

14 .

3-0

0-1

5-4

6-4

14-5-i-

29 .

6-4

19-9

16-6

Total

187

115

2-8

3-2

32-0

31-3

-19 .

19 .

-2-1

2-4

13-3

12-3

8-5-

15 .

-2-1

-7-2

-6-7

4-9

11-5-

14 .

3-8

2-0

3-3

U-5 +

26 .

0-7

1-5

11-6

13-2

Total

169

74 .

0-3

0-5

33-6

33-7

and over

14 .

-14-2

-16-9

6-7

5*7

8-5-

30 .

-0-4

6-9

11-9

13-9

11-5-

53 .

8-9

24-1

31-6

37-9

14-5 +

54 .

7-8

1-6

17-3

10-1

Total

277

. 126

151 !

2-1

5-6

67-5

Under 10

10-19 .

20 and over .

Ignition loss distributions of sods from houses in same
groups of districts where a death had occurred from :

Non-cancer cause Other cancer (i.e. not stomach)

50- 100- 125- 150- 175-200-b

8-5- .

11-5- .

14-5-1- •

Total

8-5- .

11-5- .

—

14-5-1- -

Total

8-5—

11-5- .

14-5-1- -

—

. — .

Total

All

50-

100- 125-

150-

175—

200-h

All

137

—

— .

. — .

—

132

■ 80

342

• ■

—

. —

—

105

201

119

114

208

355

P. STOCKS AND R. I. DAVIES

Tliere is an unmistakable relation between the average amount of organic
material m the soil samples taken throughout a district and the mortality there
from stomach cancer, but the regression of the latter upon the former is not a
simple one. As showui below, the association between the factors is very pro-
nounced in the part of the scale where the index lies between 2 and

Organic carbon inde.Y ... 24- 31 4i 51

jMcan stomach cancer S.M.R. . . 116 123 160 170 181
Increment of S.M.R. per unit . . 7 37 10 11

This accords with the finding in the next section that stomach cancer cases occurred
with peculiar frequency after long residence in houses where the soil contained
between 2-0 and 3-5 parts per 100 of organic carbon. Soils of the “ y? ” group
described by Davies and 'IVjume Griffith (1954) and found by them to be
associated significantly with stomach cancer deaths in Anglesey also tend to have
an organic carbon content about tliis magnitude.

Organic Content of Garden Soils Directly Associated with Cancer

Soil samples were obtained from gardens of houses where a death from stomach
cancer had occurred, and 690 of these have been analysed for organic carbon
and 700 for loss on ignition. Information as to length of residence at the house
before onset of the illness was available for 92 per cent of these and Tables II and
III show the distributions on scales of organic carbon and ignition loss for dur-
ations of under 10, 10-19 and 20 or more years, with subdivision of the districts
into 6 groups accordhig to their stomach cancer mortalitj’’ in 1947-64. The lower
parts of the tables show the corresponding distributions of alternative control series
of soils taken at houses where a death had oecurred (1) from a non-malignant cause
and (2) from non-gastric cancer. In calculating the expected frequencies for com-
parison with the stomach cancer numbers the soils were further sub-divided into
those with and vdthout ash contamination, each being calculated separately. The
aggregated result for each district group was then divided by the stomach cancer
mortality ratio to obtain the expected frequency distribution if stomach cancer
incidence were uniform and unrelated to soil characters. The divergencies
from expectation in the right hand columns of the upper parts of the tables show,
therefore, how the stomach cancer cases in excess of the normal were distributed
on the soil scales, the “ normal ” being derived in two ways, firstly from a matched
control series where cancer was not known to have occurred and secondly from the
unselected series associated with cancers of the intestine, lung, breast and a few
other sites.

In districts where the incidence of stomach cancer is below or or not greatly
above the national average (S.M.R. 50-149) a condensed comparison between the
actual and expected distributions of soils from houses where stomach cancer
occurred is shown in Table IV.

The only differences noticeable here are a concentration of the stomach cases on
soils with organic carbon exceeding 5 (31 compared with a mean expectation o
12), and a tendency for the stomach cases to occur where the soil has an igiution
Joss of 11-5 or more (110 compared with a mean expectation of 82-5). It is evident
that in the districts with not very abnormal stomach cancer inciden^ce there is a
tendency for high content of organic material in the garden soil to be associated
with occurrence of stomach cancer in the house.

SOIL AND THE CAUSATION OE CANGEU

Table IV. — Actual and Expected Organic Content of Garden Soils Associated with
Stomach Cancer in Districts with tS.M.R. Below 150

Actual cases
Exjiected /Non-cancer
from \Other cancer .

Actual cases

Expected rNon-cancer .
from \ Other cancer .

Ignition loss

Organic carbon (ptsr cent)

3 -.5-

o -4-

Total

8 -.5-

1 1 - 5-

14-5-i-

Total

Kesident less than 20 years in

bouse

199

112

210

100-4

28-2

7-0

195-0 .

120-8

40-0

19-2

20-9

200-9

103-7

25-4

5-9

195-0 .

109-0

51-8

22* 1

23-4

200-3

Resident 20 years or more in

bouse

132

120

no -0

29-3

4-0

123-9

52-2

.32-4

20-1

19-2

123-9

89-4

28-0

9-0

120-4

54-9

25- 1

14-9

25-5

120-4

In the aggregate of districts where mortality from stomach cancer is very high
(S.M.R. 150-262), if the large excess of cases over the normal expectation is
unconnected -with soil characters its distribution on the soil scales should not
differ significantlj’’ from the distribution of the expected cases, and if there is a
concentration of the excess at a particular point in the soil scale that would
support the hypothesis that a soil factor accounts for the abnormally high inci-
dence of stomach cancer in parts of North Wales, this being superimposed on the
other causes which operate throughout England and Wales. The comparison
summarised in Table V shows amongst people who had lived less than 20 j’^ears
in the house a strong concentration of the stomach cancer cases on soils -with
high organic carbon and ignition loss, similar to that found in the more normal
districts (Table W). For people who had lived 20 years or more in the house
and then died of cancer or other cause, the surplus of stomach cancer is distributed

Table V. — Actual and Expected Organic Content of Garden Soils Associated with
Stomach Cancer in Districts with High S.M.R. (150-1-)

Actual cases

Expected ("Non-cancer .

from \ Other cancer
Excess (per cent) over
mean expectation
Per cent of total

Mean expected number
Excess cases

Actual cases

Expected •
l^Other cancer
Excess (per cent) over
moan expectation
Per cent of total

Jlean expected number
Excess cases

Ignition loss

Organic carbon (per cent)

3 * o — o -(-

Total

8-5-

11-5- 14-5-i-

Total

Resident less than 20 years in house

38 29

144

146

9-1

40-9

20-3 7-0

77-3 .

19-2

16-7

20-6

23-3

79-8

15-3

28-0

25-2 9-3

77-8 .

15-2

22-3

18-3

25-2

81-0

67 256

127

15-7

44-4

29-4 10-5

100-0 .

, 21-4

24-2

30-2

100-0

7-2

38-4

23-0 31-4

100-0 .

. 24-1

16-0

13-0

46-9

100-0

Resident 20 years or more in house

52 25

149

. 14

151

6-2

27-5

43-0 7-9

84-6

. 7-3

18-1

21-4

36-7

83-5

7-4

26-0

38-2 11-3

83-5

8-3

16-1

15-2

43-9

83-5

-73

164

36 120

190

8-1

32-2

48-3 11-4

100-0 ,

9-3

20-5

21-9

48-3

100-0

-7-4

66-1

17-6 23-7

100-0 .

9-2

19-1

51-4

20-3

100-0

P. STOCKS AND E. I. DAVIES

on the soil scales in a manner very different from that expected. Two thirds
ol the excess occurs on soils with organic carbon between 2 and 3-5 per 100 com-
pared with one third of the control, and one half occur on soils ivith ignition
loss between 11-5 and 14-5 per cent compared with about one fifth of the control
IJiese differences from expectation are highly significant from a probability
standpoint, and they seem to indicate that long residence on soil of this special
type in Nortli Wales is peculiarly conducive to cancer of the stomach, the con-
nection with soils of high organic content being more pronounced where there was
appearance of stomach cancer after shorter periods of residence.

In Table VI(6) the mean organic carbon and ignition loss of soils from 43
houses where a death from stomach cancer (SC) had occurred in two Devonshire
townships are compared with a control series taken from corresponding sub-
districts of those areas The SC series gives higher mean values than the control.
(N) both for organic carbon and for ignition loss. Furthermore, as shown in Table
Vl(cr), the general level of organic matter in the soil is higher in the locality with
a high death rate from stomach cancer than in the locality with a low rate. These
results, obtained by a more rigid control method than was possible in North Wales,
agree with what has been found there.

Trace Elements in Garden Soils in North Wales, Cheshire and Devonshire Districts
tviih High and Low incidence of Stomach Cancer

Two neighbouring and similar Devonshire tomisliips A and B each with about
4,000 people, have shown over the last 10 years very different death rates from
cancer of the stomach, the S.M.R’s being estimated at 187 and 52 respectivelj'.
Owing to dissemination at A of industrial waste containing mineral substances
which could be carcinogenic, a comparison of garden soils in A and B was carried
out with the co-operation of the County Health Officers in respect of the trace
element contents, using the spectrographic method. Each place was divided
into 24 sub-areas and in each of these three gardens were chosen at random,
except that addresses where cancer was knorni to have occurred were avoided,
soil samples being taken and mixed together producing 24 samples representative
of the sub-areas in A, and likewise in B. The dried soils were analysed spectro-
graphically for 7 elements and chemically for iron and copper by the methods
described on page 20. The mean values in parts per million for the two places
are shown in Table VI(a).

The average amounts of 7 of the elements were greater in the soils from A
where the stomach cancer mortality is high than in those from B where it is low,
the excess being highly significant for cobalt, nickel and iron. Neither in
district A nor in B are the values necessarily representative of Devonsliire soils
in general, and no useful comparison with the averages for North AVales and

Cheshire in Table VII can be made. ^ ^ i <■

In the process of obtaining a control series of garden soils for trace element
analysis 36 from parts of North Wales where the incidence of stomach cancer is
abnormally high and 48 from Cheshire where it is normal were chosen from the
addresses where a person had died of a cause other than cancer after years or
more of residence in the house. Comparison of the results of spectrographic
analysis in respect of 8 elements is shown in Table VII, the method used being the
same as for the Devon soils (p. 20).

SOIL AND THE CAUSATION OE CANCER

Table VI. — Trace Elemenis and Organic MaUer in Garden Soik of
Two Devonshire Toionships

(a) Comparison between soils randomly taken from A where mort ality from
stomach cancer is high and from B where the mortality is low.

Number
in onch
place

Organic carbon (per cent) . . 72

Ignition loss (per cent) . . 72

Copper (parts per million) . 72

Zinc „ „ „ . 72

Cliromium „ „ ,, . 72

Cobalt „ „ „ . 72

Nickel „ „ „ . 72

Vanadium „ „ „ . 72

Titanium ,, „ „ . 72

Iron „ „ „ . 72

Lead (median values in p.p.m.) . 72

Mean content in

Per cent

soils from

t —

--A ^

Difference

combined

A - B

average

3-22

2-82

0-40

11-07

7-90

3-17

2-02

1-95

0-07

115

120

. -5

0-215

0-192

0-023

0-4G2

0-.32G

0-13G

1-G8

1-17

0-51

0-28G

0-3G2

. -0-07G

> • •

0-190

0-153

0-037

24-9

17-7

7-2

3-7

2-9

0-8

(6) Comparison between soils from houses where stomach cancer had
occurred (SC) and a control series from the same sub-districts of A and B
(N).

Mean content in

Number

soils of series

Ratio to

in each

DiiTerence

standard

group

SC - N

error

Organic carbon (per cent) .

3-4G

3-02

0-44

1-4

Ignition loss (per cent)

11-90

9-9G

1-94

1-7

Copper (parts per million)

2-68

2-18

0-50

1-5

Zinc ,, jj „

181

103

2-8

Chromium „ „ „

0-289

0-193

0-09G

2-8

Cobalt „ „ „

0-502

0-371

0-131

2-5

Nickel „ „ „

1-93

1-48

0-45

2-2

Vanadium „ „ „

0-407

0-303

0-104

2-0

Titanium ,, ,, ,,

0-132

0-170

. -0-038

Iron „ „ „

19-5

21-8

. -2-3

Lead (median values in p.p.m.) .

2-05

3-50

. -1-45

Table VII. — Trace Element Content of Garden Soils not Directly
Connected with Cancer in NoHh Wales and Cheshire

Zinc

Chromium

Cobalt

Nickel

Titanium (soluble) .

Vanadium

Iron

Lead (median values)

Mean content

(p.p.m.) in

North Wales Cheshire

(36)

51-2

0-230

0- 522

1- 046
0-228
0-342

23-8

2 - 1

(48)

54-3

0-178

0- 528

1- 230
0-213
0-455

20-7

5-2

Difference

Ratio to
its standard
error

-3-1

0-2

+ 0-052

2-0

-0-006

0-1

-0-184

1-8

+ 0-015

0-7

-0-113

2-1

+ 3-1

1-2

-3-1

P. STOCKS AIW R. I. DAISIES

TJie Noi’fcJi "Wales soils slioiv an excess of chromium, wliilst nickel A^anadium
and lead are more p entiful in the Chesliire soils. There is no indicat^n from theS
comparisons that the general leAml of zinc is any greater in districts Avith high
incidence of caiiTOr of the stomach than it is elseAvhere, and for cobalt this Ayas
It Devonshire toAvnsliips investigated but not in the Cheshire and

North ales area. This has to be kept in mind Aidien seeking an explantion of
the results recorded m the next section. The excess of chromium, cobalt and
nickel m locality A (Table Via) may be of industrial origin and inimstigation of
this possibility is not yet complete.

Tictce Elemtiiis in Garden Soils Directly Associated with Cancer

In the Devonshire toAvnship A, already referred to, 37 deaths from cancer
of the stomach had occurred in 15 years, and soil samples Avere taken from the
gardens of houses Aidiere the deceased persons had last lived, the number of years
residence there before death being ascertained AA'hen possible. Within toAimship
B 7 such deaths had occurred and samples Avere taken similarly. Each of the
44 cases AA’as matched by a non-cancer sample from the same sub-area, and
Table VI{6) compares the mean trace element contents of the soils in the stomach
cancer (SC) and non-cancer (N) series.

In North Wales spectrographic examination has been made of 73 soils from
houses in the 5 counties Aidiere a resident had died of stomach cancer after living
there 10 years or more, and of 39 soils similarly associated Aidth a death from cancer
of the intestine, lung or breast, the frequencies of these being 29, 7 and 3 respec-
tivelJ^ From the Cheshire areas, comprising the Wirral, Runcorn, Ej'^mm and
the rural districts of Chester and TarA’'in, 31 soils associated Avith stomach cancer
and 9 associated AA'ith intestinal cancer haAm been analysed. The mean trace
element contents of these groups (SC and OC) are compared in Table VIII Avith
those for the non-cancer control series (N).

Zinc. — In North Wales the stomach cancer soils shoAv a Avide range of A^alues
from 4 to 441 part per million, noth a mean of 81-2 compared Avith 51-2 for the
non-cancer controls, the excess of 30-0 being tAvice its standard error. In Cheshire
the SC soils shoAV a range from 3 to 387 Avith a mean of 83-4 compared Arith 34-3
for the controls, the excess of 29-1 being 1^ times its standard error. In the
combined area the excess of 28-8 is 2-4 times its standard error. In the tAVo
Devon localities AA^here the zinc IcAmls happen to be higher the mean for the SC
soils is 181 compared Avith 103 for the matched controls, giving an excess 2-8
times its standard error.

The odds against finding such agreement in three independent series by chance,
AAuth t = 2-0, 1-5 and 2-8, are enormously great, and it must be concluded that a
zinc content of the soil higher than the local average is a factor favourable to the
appearance of stomach cancer, and that this is not confined to districts where the
general incidence is specially high. Indeed, as shoAra in Tables VI and VII,
there is no tendency for districts AAuth high stomach cancer mortality to haAm
higher zinc levels in the soils as a Avhole than districts AAuth Ioaa^ mortality, and
yet in the gardens of houses Avhere deaths from stomach cancer occurred the zinc
level is higher than in other gardens of the same area. This seems to indicate
that another factor must be present Avhich acts in conjunction AA’ith zinc and Avhicli is
more plentiful in soils of districts Avhere stomach cancer incidence is high than

SOIL AND THE CAUSATION OF CANCER

Table VIII. — Qiiaiitities of Trace. Elements (parts per million) in Soils from
Gardens of Houses in North Wales and Cheshire where a Death had Occurred
from Cancer of the Stomach (SC), Other Cancer (OC) or from a Non-camcer
cause (N)

North Wnics* Chcsliirc* Combined area*

Mean

Diff. ’

Mean

Diff. ’

Mean

Diff.

•

No.

(l).p.m.)

(C - N)

No.

(p.ji.m.)

(C - N)

(p.p.m.)

(C - N)

S.E

Zinc .

.-)l-2

.54-3

53-0

, ,

. ,

Sl-2

-f-30-0

83-4

+ 29-1

81-8

+ 28-8

12-0

04-9

-M3-7

.50- 1

+ 1-8

03-2

+ 10-2

1 1 -8

Cobalt

. N

0-o22

0-528

0-525

0- Of).!

-f0-i33

0-025

+ 0-097

0-040

+ 0-121

0 049

0-.7S3

-f-0-001

0-522

-0-000

0-572

+ 0-047

Nickel

. N

1-040

1-230

1-151

1-010

-0-030

1-412

+ 0-182

1-134

-0-017

0-921

-0- 125

1-381

+ 0-151

1-007

-0-144

Chromium

. N

0-230

0- 178

0-200

0- 31.-1

0-085

0-232

-^0-054

0-291

+ 0-091

0-020

0-290

■+-0-000

0-154

-0-024

0-205

+ 0-005

0-020

Vanadium

. N

0-342

0-4.55

0-407

O- 3 .- 1 O

J-0-6i4

0-4.53

-0-002

0-385

-0-022

0-097

0-300

-f 0-024

0-431

-0-024

0-378

-0-029

Titanium

. N

0-228

0-212

0-219

0-202

-o’oio

0-258

+ 0-045

0-219

Nil

0-238

-bO-OlO

0-102

-0-051

0-224

+ 0-005

Iron .

. N

23-8

20-7

22-1

21-2

-2-0

29-5

-i-8-8

23-7

+ 1-6

24-3

-rO-5

21-9

-1-2

23-8

+ 1-7

Over 5 p.p.m.

% over 5 p.p.m.

^ A ^

No.

No. % '

No.

No. % '

Median % C — N

Lead .

. N .

33-3

52-1

4-75

44-0

SC .

42-5

58-1

4-05

47-1

+ 3-1

OC .

51-3

77-8

5-00

59-2

+ 15-;

* Garden soils where a death had occurred after 10 years of more of residence at the house.

Diff.” means the difference between the mean for the cancer group (C) and that for the control
(N) group. “ S.E.” = standard error.

where it is low. In the Devonshire locality A this might be present in the indus-
trial waste which has found its way in the past into many of the gardens but which
would only become important in regard to stomach cancer where there was also
3^ high zinc level, and study of this problem is continuing and will be reported
upon in another paper.

In the Cheshire and North Wales region, amongst the soils associated with
stomach cancer those taken from ground where vegetables or fruit were being
^own showed a zinc distribution somewhat different from other garden sods, as
s own m Table IX, whereas no such difference appears in the control series.

le organic carbon content failed to show any differences between vegetable
garden and other sod. Since zinc is an active part of some enzyme systems and is
2

P. STOCKS AND R, I. DAVIES

Table IX. Zino and Cobalt Content of Soils from Vegetable or Fruit

Compared with Other Garden Soils

Growing Ground

Zinc

r“" — — — ^

0- 20- 50- 80- 140+ Total

10 19 14 8 5 56

3 8 5 4 13 33 .

5-9 11-2 8-2 4-7 3-0 33 ,

■// = 14-8, n = 3,P < 0-01

Control . . V or F . 12 16 9 3 6 46

Other . 9 12 5 6 4 36

Exp.» . 9-4 12-5 7-1 2-3 6-7 36 .

■F = 0-77, n = 3,P > 0-8
* Distribution o.vpected from that of tho V-P series.

Part of

Series garden

Stomach cancer . V or F
Otlier
Exp.*

Cobalt

r- —

0-1- 0-4- 0-60- 0-8+ Total

6 56

11 33

9-4

11-8

8-2

3-6 33

2-38, n

= 2,

P = 0-3

5 46

4 36

12-5

7-1

3-9 36

;y* = 2-45,« = 2, P = 0-3

concerned ivitli plant life and the processes of gastric digestion a connection
between stomach cancer incidence and the amount present in soil is by no means
wildly improbable.

Table shows that in North Wales the average zinc content of 39 soils
associated with other cancer is rather greater than the control figure, but for the
29 intestinal cases included the mean is 53-0, differing inappreciably from the
control, and this is true also of the Cheshire cases which are all intestinal, so the
excess is confined to the 7 lung cancer cases and is not statistically significant.

Cobalt . — In North Wales the SC soils show a range from 0-17 to 2-80 parts per
million with mean value 0-655, and the N soils show a range from 0-18 to 1-04
with mean 0-522, the stomach cancer excess being twice its standard error. In
Cheshire the SC range is from 0-21 to 1-55 with mean 0-625, compared with the
control mean 0-528, an excess of 0-097 (t = 1-1). The combined area gives an
excess of 0-121 and the Devonshire data an excess of 0-131, each of these being
2| times their standard errors. The odds against such a result arising by chance
are ver}'’ great, and it must be concluded that a high cobalt level in the soil is
favourable to a higher incidence of cancer of the stomach but in view of Table
ITII, this seems to depend upon conjunction with some other substance, as for
zinc. The other cancer series shows no significant difference from the control.
Table IX shows no significant differences between the cobalt content of vegetable
garden and other garden soils. The element is known, however, to be concerned
in plant and animal economy and also to have carcinogenic properties, so the
statistical connection between a high soil content and stomach cancer incidence
is deserving of further study.

AGcM.—ln North Wales the SC soils have contents ranging from 0-30 to
2-62 parts per million, those from Merionethshire having specially high values,
and the mean of 1-016 is slightly less than that of the controls whose range is from
0-41 to 2-48. Amongst Cheshire SC soils only 29 per cent have values below 1
compared with 58 per cent in the Welsh series and the mean 1-412 exceeds the
control figure by 0-182 (t = 1-6). In the Devonshire localities the nickel levels
were still higher and the SC mean exceeds the N mean by 0-46 (t = 2-2). It is
doubtful whether these differences indicate a connection with stomach can^r
since the correlation is if anything negative in the combined Cheshire-Aorth
Wales region. Comparison between the OC and N soils shows a similar

SOIL ^VKD THE CAUSATIOX OF CANCER

discrepancy between the two parts of this region, neitlier of tlie differences being
statistically significant.

Chromium.— The A*ariation in content is smaller for this element, the range in
SC soils being from 0‘06 to 0‘92 and in the controls from 0‘05 to O' 1 1. In I^orth
Wales the SC mean of 0-315 exceeds the control mean by 0-085 which is highly
significant (t = 3-1) ; and in Cheshire there is likemse an excess of 0-054 (t = 1-5).
In the combined area the stomach cancer excess is 4 times its standard error and
the Devon comparison gives a similar excess of 0-096 (t = 2-8). When median
chromium values based on more than one S.C. and control soils were compared
in 17 separate districts, the S.C. median exceeded the control median in 11 ; but
as Table X shows, the surplus incidence occurs not at the highest chromium
levels but where the content hes between 0-3 and 0-6 parts per million (33 out of
89 instead of 5-5 expected).

The North Wales soils show also for the OC series, winch are mostlj’- intestinal
cancer, a mean value which is 0-060 above that of the controls and in the com-
bined area the excess of 0-065 is statisticallj’^ significant. In Table X the 3 ’^ show a
concentration between 0-3 and 0-6 and for both the stomach and other cancer
soils the ;\;2test gives P < 0-001. The association -with cancer seems to differ from
that of zinc and cobalt in that (1) it applies to intestinal as well as to stomach
cancer and (2) since chromium levels tend to be higher in all soils in districts w^here
the incidence of stomach cancer is high it is not necessarj’' to assume that
chromium acts onlj'’ in conjunctioir vith some other substance.

Table X . — Chromium Content of Garden, Soils Directly Associated with Cancer

Compared with Controls

Chromium (parts per million)

Stomach cancer

— *

Series

S.1I.R.

0-2-

0-3-

0-4-

0-6-f

Total

Non-cancer ,

Under 150

150 and over

3 !

Stomach cancer

Under 150

150 and over

Total

Expected*

48-0

30-5

3-8

1-7

5-0

Other cancer

Under 150

150 and over

Total

Expected*

•

26-4

15-6

2-0

1-0

3-0 .

* Expected from the control, given the same weighting according to S.M.R. of district.

Vanadium. — The Devonshire data show a significant excess of 0-104 (t = 2-0)
when the mean vanachurn content of the SC series of soils is compared vdth the
matched control, but indications from the other areas are not clear. The means
are greatty affected by occasional soils with very high amounts of the element
tor example m North Wales the N series includes one with 6-20 p.p.m. the next
highest value being 0-80, and in Cheshire N soils the highest values were 3-44 foUowed

fL d ^ dififtculty, reveals a great difference between

the distributions of N soils in the groups of districts with high and low stomach

P. STOCKS AND R. I. DAVIES

cancer mortality, two tliirds of the soils in the former group having less than 0-2
p.p m compared with one eighth in the latter. When the SC soilslre compared
vith tlie distribution expected from controls within the same district groups the
^flFerence IS hardly significant = 9-1, n = 4, P = 0-06), and a Xefee of
the same kind IS seen for other cancer, namely an excess of soils ivith content
around 0-3 parts per million. No definite conclusions can be drawn, but there is a
curious resemblance to the cliromium comparisons in Table X.

Table XI. Vanadium Content of Garden Soils Directly Associated with Cancer

.1 •.1 ^ , 1 ^

Compared

with Controls

Stomach cancer

Vanadium (parts per million)

/ .

Series

S.M.R.

0-2-

0-4-

0-6-

0-8-f

Total

Non-enncer .

Under 160

150 and over

Stomnch cnncer

Under 150

150 and over

Total

Expected*

39-1

20-7

17-0

6-0

6-2 .

Other cancer

Under 150

150 and over

Total

Expected*

22-9

10-8

8-1

2-9

3-3 .

Expected from the

control, given the same weighting according to S.M.R. of district.

Titanium. — The analyses relate to the titanium extractable by the standard
solvent used in preparing the soil for spectrograpliic study, the insoluble forms
sueh as rutile which are plentiful in soil not being thought likely to have any
biological activity. In none of the areas is there anj’^ indication of any connection
with cancer.

Iron. — In Devonsliire and North Wales the stomach cancer soils do not
differ significantly in average content from the controls, the mean levels being if
anything below expectation. In Cheshire, however, the SC series shows an excess
of 8-8 parts per million (t = 3-0). No appreciable differences appear for other
cancer.

Lead . — Since the quantitative assessment of amounts of this element by the
spectrographic method is difficult when the level exceeds 5 parts per million,
the statistical comparison has been made by comparing proportions of the total
soils having 5 or more p.p.m., and by the median values, these measures being
unaffected by uncertainties as to the exact values in the upper part of the scale.
The Devonshire data show a lower median for the stomach cancer series than for
the controls, and 23 per cent of each series had 5 or more parts per million of
lead. In the Cheshire-North Wales area there was no significant difference by
either measure, but soils connected ■with non-gastric cancer show greater pro-
portions ■with a high lead content than the controls, this series consisting mainl}’’

of intestinal cancers. j- ^ • i.

Copper . — The amounts of copper in the soils from the two De^^onshire distncts
were determined by a colorimetric process in a separate acetic acid extract of the

SOIL AKII THE CAUSATION OF CANCER

soil, as described below. Table VI(b) shows that the mean eontcnt of the soils
from houses where stomach cancer had occurred is higher by 0-50 p.p.m. than that
of the controls. The ratios of cobalt to copper are about 0-18 m each group,
and the ratios of nickel to copper arc 0-7 in each group, giving no indication that
copper has a counteracting effect. The ratio of zinc to copper, however, is 0-8
in the stomach cancer soils compared with 4-7 in the controls.

SUMMARY

Chemical and spectrograpliic study of garden soils in North Wales, Cheshire
and two localities in Devonshire has established correlations between the amounts
of certain constituents and the frequenej- of cancer of the stomach. Organic
matter, zme and cobalt are related positively and significantly with stomach
cancer incidence but not ivith intestinal cancer, whilst chromium is connected
vdth the incidence of each of these. The abnormal rates of stomach cancer in parts
of North Wales are associated vnth long residence on soils whose organic content
lies between definite limits. Soil rich in zinc or cobalt is found n-ith excessive
fi-equenej’- where a case of stomach cancer has occurred but the geographical
distribution of such soils appears to be unrelated to that of stomach cancer rates.
Vanadium and iron show inconclusive relations with stomach cancer in one of the
areas, whilst nickel, titanium and lead show no connection anjnvliere udth this
form of cancer.

METHODS OF TRACE ELEJIEXT ANALYSIS

Sods were examined for elements likely to be taken up by plants in micro- or
trace quantity. The likelihood of this uptake is to some degree related not to the
total quantity of each element in the soil, but rather to a combination of quantity
weatherabUity and ease of solubility. To simulate tins “ availability for plants ”
it is customary to extract soils with very dilute acetic acid or neutral salt solutions
or even with dilute solutions of ion complexing agents. For the present investiga-
tion N/2 acetic acid was chosen (20 g. soil/800 acid) the acid being in contact
with the soil with occasional shaking for 12 hours. Analysis of the acetic acid
extract then followed closely the procedure of llDtchell (1945). This involves a
separation of the micro-elements from those present in large amounts (e.g. K,
Ca, Na, Mg) resulting in a concentrate of the micro-elements. The precipitate of
all these elements is ultimately arced by direct current, the arc light being
examined by a Bulger Large Quartz Spectrograph.

In the present investigation 30 mg. AlgOg, 2-5 mg. FogOg and 0-4 mg. Cd.
were introduced before precipitation to ensure consistent arcing and to provide
elements for reference in the spectrogram. Cobalt, Nickel, Chromium, Vanadium,
Titanium, Lead and Zinc were then determined quantitatively on the spectrograrn
using Iron, already found by chemical analysis, as a reference standard. Zinc was
also determined by using the added Cadmium as reference, and it is this second
value which has been used throughout the present work.

Available copper cannot be determined spectrographically because of limita-
tions imposed by contamination. It was necessary therefore to devise a separate
method, an adaptation of the colorimetric estimation by Zinc Dibenzyldithio-
carbamate (Andrus, 1955). For this a fresh extract was prepared (20 g soil bv
800 ml. A /2 acetic acid). The extract was evaporated to dryness, oxidized with

F. STOCKS AND R. I. DAVIES

a little nitric acid and dissolved in N. acetic acid. This was shaken with a
solution of Zince Dibenzjddithiocarbamate in carbon tetrachloride and the
intensity of colour produced in the latter by copper was estimated with a spectro-
photometer.

We are greatly indebted to the Medical Officers of Health and their Assistants
in Cheshire, the 5 counties of North Wales and the 2 districts of Devonshire for
the collecting of soil samples and for suppljdng the information as to deaths from
cancer and other causes. Our thanks are due also to the British Empire Cancer
Campaign for supporting the Avork and to the Analj'sts and Technicians in the
Department of Agricultural Chemistry at Bangor whose sendees har^e been
provided by the Campaign.

DEFERENCES

Andrus, S. — (1955) Analyst, 80, 514.

Daat^es, R. I. AND WvNNE GRIFFITH, G. — (1954) Brit. J. Cancer, 8, 56, 594.

Huxley, J. — (1958) ‘ Biological Aspects of Cancer ’, London (Allen & Unwin),
j\IiTCHBLL, R. L. — (1945) ‘ SpcctrographicAnalj'sis of Soils, Plants and related Materials ’,
Technical Communication 44. Harpenden (Commonwealth Bureau of Soil
SciGIlCC) •

Stocks, P. — (1955) Bep. Brit. Emp. Cancer Campgn, 33, 468. — (1956) Ibid., 34, 520. —
(195Sn) Ibid., 36, 342. — (1957) Ibid., 35, Supplement, p. 95. — (19586) In ‘ Cancer ’,
London, (Butterworth), Chapter 4, Vol. 3, p. 153.

WaXiKLEY, a. and Black, I. A. — (1934) Soil Sci., 37, 29.

SECONDARY TUMOURS OF THE HEART

W. J. HANBURY

From the Department of Pathology, St. Bartholomeiu's Hospital, London, E.C.l

Received for publication December 30, 1959

The incidence of metastatic tumours in the myocardium was found to be 5
per cent in a series of 500 cancer necropsies reported by Willis (1952), who
commented that the supposed infrequency of secondary growths in the heart
wall was due to inadequate observation. Among the more recent papers on the
subject are those of De Loach and Haynes (1953), Burnett and Sliimkin (1954)
and Goudie (1955).

The relative susceptibilities of different tissues to blood-borne metastases
constitute one of the many interesting problems of cancer, and the recording of
metastases in a collected series of cases can still serve a useful purpose.

The present study is based on the pathology of 50 cases of discrete secondary
tumours of the heart. A few of these are comparatively recent, while the rest
were found in the records of this hospital. Factors to be considered are the dis-
tribution of tumours within the heart, the types of associated primary tumours,
and the incidence of associated metastases in other organs. In addition, one of
the 50 cases will be presented in more detail, being of particular interest with
regard to the “ soil ” hypothesis of tumour metastases (Willis, 1952).

The total incidence of secondary cardiac tumours cannot be given for this
series, as the cases were not taken from a consecutive period over which the
records are complete. Of the 50 cases, 28 were in males and 22 in females. Cases
of leukaemia and of Hodgkin’s disease have been excluded, as also have tumours
directly invading the heart from adjacent structures. Metastases invohdng the
endocardium and epicardium have been included, but those involving the parietal
pericardium only have been excluded. The finding of neoplastic cells within
lymphatic vessels of the heart has not been regarded as constituting true
metastatic growth.

Table I. — Distribution of Cardiac Metastases

Part of heart involved

No. of cases

Right atrium

Right ventricle

Left atrium .

Left ventricle

Interatrial septum .

Interventricular septum 's

Epicardium .

Epicardium only

Myocardium .

Endocardium

W. J. HANBURY

DISTRIBUTION OP TUBIOURS WITHIN THE HEART

secondary tumours Avithin the heart is shoirn in Table T
The metastases were solitary in 14 cases and multiple in 36 casls Tit il J'
shous a shght preponderance of tumours on the rieht side nf the h^a i ^
ventricles to be more frequently affected ^ ^

“ I Haynes, 1953) has been variable, but Willis (1952) concluded that

differmiTntrts are -JffeK equally prone to metastasis, and that the

aitterent parts are affected proportionately to their bulk The table also shows

a loir incidence of septal involvement, but tliis is probably due to the septa Lt
being specifically mentioned in reports on cases udth multiple metastases^

Table 11.— Primary Tummirs with Cardiac Metastases

Primary tumour N^

Carcinoma .... (33)

Bronchus . . . . jO

Skin ..... 4

Kidney .... 4

Oesophagus ... 3

Stomach .... 2

Carvis uteri ... 2

Breast .... ]

Tliyroid .... 1

Pancreas .... 1

Rectum .... 1

Vulva .... 1

Testis .... 1

Unknown origin ... 2

Malignant melanoma . . 5

Retieulosarcoma ... 4

Ljmphosarcoma ... 1

jMultipIe myeloma ... 1

Fibrosarcomo (breast) . . 1

Osteogenic sarcoma (tibia) . 1

Haemangio.endothelioma (liver) 1

Chorionepitheliomn (uterus) . 1

Teratoma (testis) ... 1

Neuroblastoma (adrenal) . 1

Total . 50

TYPES OF PRIMARY TUMOURS ASSOCIATED RUTH CARDIAC METASTASES

The imrious sites and types of the primary tumours which produced cardiac
metastases are shoivn in Table II. The figures indicate no striking differences
from those of other reported series (reidewed by De Loach and Haynes, 1953),
the relatii^ely liigh incidence of metastases from carcinoma of the bronchus and

EXPLANATION OF PLATE.

Fio. 1. — The heart sectioned to show multiple corcinomatous metastases in the left atrium and
left ventricle. The arrow indicates calcification of the base of the posterior cusp of the mitral
valve.

Pin. 2. — The heart sectioned to show metastases in the right ventricle as well as on the epicardial
surface of the left ventricle.

Fig. 3. — Photomicrograph showing infiltration of left ventricular myocardium by squamous
cell carcinoma. H. and E. x 85.

Hanbuiy.

SECONDARY TUMOURS OF THE HE^VRT

from malignant melanoma and reticulosarcoma being typical. The proportion of
cases of skin cancer is somewhat higher than usual, and there is a lower incidence
of carcinoma of the breast. Of the 33 cases of carcinoma, 15 are squamous-celled,
11 are adenocarcinomas and seven undifferentiated.

METASTASES ASSOCDVTED WITH SECONDARY TUMOURS OF THE HEART

In most of the reported cases of secondary cardiac tumours there have been
widespread metastases in many other organs, although these have not usually
been listed in detail, and in particular there has been a high incidence of associated
malignant involvement of other intrathoracic structures (Lymbumer, 1934 ;
De Loach and Hajmes, 1953). The tumours were also mdely disseminated in the
majority of cases in the present series, but in seven of the 50 cases there was no
other apparent primarj^ or secondary intrathoracic neoplastic involvement, and
in two cases there was no definite information on this point.

The incidence of associated metastases in other organs is shorni in Table III.
Care was taken to exclude instances of direct neoplastic extension or lymphatic
permeation as far as possible. For this reason secondary growths of the pleurae,
peritoneum and skin have been excluded from the table, as also have lymph node
metastases and serosal deposits on the abdominal organs.

Table III . — Metastases associated with Secondary Tumours of the Heart

No. of cases

Percentage of

Percentage of
metastases in

with metastatic

total coses

Willis’ 500 Cancer

Organ

involvement

(50)

necropsies

Liver

Kidneys

7-0

Lungs

Bones

13-6

Adrenals

Intestines .

Spleen

Pancreas

Thyroid

Stomach

0-4

Brain

—

Ovaries

—

Oesophagus

—

Urinary bladder .

—

Skeletal muscles .

—

Gall-bladder

Meninges .

—

Breast

Tongue

Tonsil

Testis

Subgluteal bursa

—

As metastatic tumours of the heart are usually accompanied by widespread
metastases in other organs, the incidence of the latter should be higher than in
unselected cases of malignant disease. The relatively high incidence of cardiac
metastases from bronchogenic carcinoma woidd also tend to raise the frequency
of secondary growths in such organs as the adrenals, kidneys and bones. In Table
ill are shown, for comparison, the percentages of metastases in certain organs

W. J. HANBURY

found by illis (1952) in his 500 consecutive cancer necropsies. From tiiis com-
parison it can be seen that in the present series there are particularly high per-
centages of associated metastases in the intestines, spleen, pancreas, thyroid and
stomach. Of these organs the spleen is perhaps of most interest, as it was com-
parativelj^ easj'" to check that the metastases were truly blood-borne and within
the splenic substance. It is possible tliat some common factor exists to make
such organs as the heart and spleen more susceptible “ soils ” for metastases in
certain cases, these organs normally being relatively free of secondary tumours.
Of the eight cases of associated metastases in the spleen there were four carci-
nomas, two melanomas, one fibrosarcoma and one haemangio-endothelioma.

In Ljnnburner’s (1934) series of 52 cases of secondary cardiac tumours there
were seven instances (13'5 per cent) of associated splenic metastases, a similarly
high figure ; six of these were carcinomas and one a sarcoma. Eitchie (1941) also
reported one sarcomatous and two carcinomatous splenic metastases from 16
cases of metastatic tumours of the myocardium.

The following case illustrates an unusual distribution of secondary growths,
with involvement of the heart and spleen.

CASE REPORT

H.H., a woman aged 63, had a radical vulvectomy for carcinoma of the vulva
in January, 1957 at this hospital. Two right inguinal lymph nodes showed neo-
plastic infiltration, the tumour being a poorly differentiated squamous cell carci-
cinoma. In April, 1958 the patient was admitted to another hospital with pyrexia,
a skin rasli and joint pains. Rheumatoid artliritis and erjdhema nodosum were
diagnosed, and there was a good response to steroid therapy. Four months later,
however, the patient became generally unwell and somewhat disorientated, and
was re-admitted to tin's hospital on August 23rd. There was a past history of
rheumatic fever at the age of 16 and alopecia totalis for 20 years. There had been
eight pregnancies, mcluding three miscarriages.

On examination there was no fever, but the patient was found to have auricular
fibrillation, a slightly raised venous pressure, slight left ventricular hypertrophy,
a systolic murmur indicating mitral incompetence, and Cheyne-Stokes respiration.
The E.S.R. was 14 mm. in 1 hour (Westergren), and an electrocardiogram con-
firmed the auricular fibrillation and was reported to show left , ventricular
ischaemic changes. There was also a swollen right leg. Generalised carcinomatosis
was suspected, and the patient died on the day after admission.

At autopsy there were metastases of squamous cell carcinoma in the abdominal
lymph nodes, liver, spleen, peritoneum and heart, but none was found in any
other intratlioracio structure or in the brain. Recent ante-mortem tlirombi were
present in the splenic and right femoral veins. . , , j. u

The heart weighed 470 g. and showed moderate left ventricular hypertrophy.
Very numerous wliitish metastases (measuring up to 0-8 cm. in diameter) were
present in the epicardium, myrncardium and endocardium of all four chambers,
(Fig. 1 and 2) including the interatrial and interventricular septa. ®

mitral ring and base of the posterior cusp of the mitral valve showed a zone o
calcification measuring 1 x 0-5 x 0-3 cm., which made the mitral orifice some-
what rigid. The remainder of the valve and the chordae tendmeae showed no
fibrous thickening, and the other valves were normal. The coronary arteries showed

SECONDiVKY TUMOURS OE THE HEART

slight atheroma but were not appreciably narrowed, and the aorta was only
moderately atherosclerotic.

INIicroscopic examination shows extensive infiltration of tlie heart wall by
moderately well differentiated squamous cell carcinoma (Fig. 3) vdth some kera-
tinization and cell-nest formation. In the myocardium columns of tumour cells
extend between the muscle fibres and are often closelj’’ related to the small blood
vessels. There is a moderate degree of reactive fibrosis with associated chronic
inflammatory cell infiltrations. No evidence of active rheumatism can be seen.
The calcified area at the base of the mitral valve contains no carcinoma cells and
is surrounded bj’^ dense fibrous tissue with a minimal inflammatory reaction.

The spleen weighed 245 g. and contained multiple metastases measuring up
to 1 cm. in diameter, klicroscopically, these are composed of fairly well differen-
tiated squamous cell carcinoma with cell-nest formation. Several small arteries
and veins contain recent ante-mortem tlrrombi.

The main interest in tliis case lies in the very extensive cardiac metastases in
the absence of other intrathoracic metastases, together with the associated rheu-
matic history. Although the heart showed no evidence of active rheumatism or
of extensive rheumatic scarring, the partial calcification of the mitral valve was
probably rheumatic in origin. It is possible that the heart may have become more
susceptible to metastasis on account of the previous rheumatism, but this inter-
pretation can only be hjq)othetical, and the exact nature of such a susceptibility
can only be conjectural at present.

A history of rheumatic fever was also found in two other cases of this series,
in one of wBch there was scarring of the mitral valve. In two cases there was a
history of scarlet fever. One of the cases reported by Scott and Garvin (1939)
also had rheumatic heart disease.

SUMMARY

A study has been made of the pathology of 50 cases of discrete secondary
tumours of the heart. Factors considered were the distribution of tumours within
the heart, the types of associated primary tumours, and the incidence of asso-
ciated metastases in other organs. One case with a rheumatic history, carcinoma
of the vulva, very extensive cardiac metastases and no other intrathoracic neo-
plastic involvement, is described in more detail.

I vnsh to thank Professor J. W. S. Blacklock for helpful adduce, ]\Ir. J. W.
Miller for histological sections, Mr. N. K. Harrison for the photo^aphs, and
Dr. G. S. Sansom for the photomicrograph.

REFERENCES

Burnett, R. C. and Sheukin, M. B. — (1954) Arch, intern. Med., 93 205
De Loach, J. F. and BLaynes, J. W. — (1953) Ibid., 91, 224.

Goudee, R. B. — (1955) Brit. Heart J., 17, 183.

Latmbuener, R. il.— (1934) Canad. med. Ass. J., 30, 368
Ritchie, G.— (1941) Amer. J. Path., 17, 483.

Scott, R. W. and Garvin, C. F.— (1939) Amer. Heart J., 17 431.

WiLLis^,;^.^ A^— G9^52) ‘ The Spread of Tumours in the Human Body.’ 2nd ed. London

Yatee, W. M.— (1931) Arch, intern. Med., 48, 627.

EFFECT OF THIO-TEPA ON ADVANCED
J\IALIGNANT OVARIAN TmiOURS

0. ENGLANDER and A. SARANGI
From the Centre of Radiotherapy, Leicester Royal Infirmary

Received for publicntion December 8, 1959

Treatment with Thio-TEPA has produced, in a significant number of patients
suffering from very advanced carcinoma of ovary, marked temporarjr retrogres-
sion of growtli and improvement of symptoms ; in a few cases temporary clinical
disappearance of growth, ascites and disappearance of symptoms was acliieved,
and those patients resumed their normal activities enjoying life for the duration
of control of groudh. Our article contains obsen^ations made from January
1957 till June 1959, and an attempt of quantitative presentation of clinical
results was made. Our observations confirm and emphasise previous reports.

Triethylene thiophosphoramide (Tliio-TEPA) is an alkylating agent struc-
turally related to nitrogen mustard and triethjdene melamine and has a similar
cytotoxic effect. The clinical effect appears to differ significantly.

Thio-TEPA was synthesised in the Lederie Laboratories.

Shay ei al. (1953) reported marked improvement in some cases of chronic
leukaemia and of Hodgldn’s disease which were treated udth Thio-TEPA ; thej’-
also reported an encouraging response in two patients sufi^ering from metastases
from mammary cancer.

These authors quoted and reviewed briefly the extensive experimental work
of the Lederie group of workers and described their own work which led to the
clinical trial.

Bateman published a detailed report on the clinical effect of Thio-TEPA in
99 cases of advanced malignant solid tumours (Bateman, 1955), and on 380
cases (Bateman, 1968).

Leonard, Israels and Wilkinson (1956) reported the effect of Thio-TEPA on
Hodgkin’s granuloma, chronic lymphatic leukaemia, polycjdihaemia and other
reticuloses.

Many valuable reports have been published since 1953. A monograph of
1266 pages containing a series of papers on the chemistry, biological effects and
clinical results of alkylating agents was published in April 1958 by the New York
Academy of Science. This monograph also contains studies on the effect of
Thio-TEPA on malignant disease and on haemopoiesis by Wright, Golomb and
Gumport (1958), Ultman, Hayman and Gelhorn (1958), Olson (1958), Shay and
Sun (1958), Bateman (1958), Alpert (1958), Moore (1958), Leone (1958) and other
related papers with references to previous work and to other authors.

The effect of Thio-TEPA on Hodgkin’s disease, chronic lymphatic leukaemia
and other reticuloses has been reported to be similar, but not superior, to that

of nitrogen mustard. j

A few cases of various groups of solid malignant tumours have been treated
with Thio-TEPA. Only in a verj’- few of these has temporary effect of varying

EFFECT OF THIO-TEPA ON OVARIAN TUMOURS

degree and duration been reported. Further systematic observation and collec-
tion of data is necessary.

Reports have repeatedly been published on marked temporary regression of
tumour and decrease of ascites, with improvement of symptoms, in advanced
carcinoma of the ovary, confirming Bateman’s original observations published
first in 1955. Bateman also observed in a large proportion of cases of carcinoma
of breast with secondaries, temporary regression of tumour masses, healing of
tdceration, recalcification of bone lesions and control of effusions.

Recently Watson and Tunrer (1959) reported the favourable response of
breast cancer to combined therapy with Thio-TEPA and testosterone propionate.

Tliio-TEPA has a marked toxic effect on haemopoietic tissue which results in
reduction of the number of leucocytes and of platelets ; excessive doses ^vill lead
to irreversible changes of haemopoietic tissue, which may be fatal. Haemato-
logical control during treatment is therefore essential. In therapeutic doses the
effect on haemopoiesis is reversible on cessation of treatment. Compared with
nitrogen mustard the side effects, such as nausea and vomiting, are very slight
and in most patients absent. Thio-TEPA has been injected intra-venously,
intra-muscularly, into the tumour, into pleural or peritoneal cavity if fluid was
present, and intra-arterially for regional treatment ; it has also, though rarely,
been given by mouth.

Bateman (1955) emphasized that Thio-TEPA was most effective when injected
directly into the tumour.

We have treated 33 cases of malignant disease -with Thio-TEPA :

Group A — carcinoma ovary — 17 cases.

Group B — carcinoma breast — 9 cases.

Group C — miscellaneous — 8 cases (reticulum cell sarcoma 1, Brill
Symmer’s 1, carcinoma cervix 1, carcinoma bronchus 2, post cricoid
carcinoma 1, myxosarcoma 1, secondary adenocarcinoma of groin of
unknown primary -f-? secondaries in lungs).

Our observations on group B and C will be evaluated separately.

We present here the observations on the effect of Thio-TEPA in 17 cases of
advanced carcinoma of ovary, which were treated in this department from
January 195/ till June 1959. During those 29 months 63 cases were referred to
the Radiotherapy Department and 52 cases were treated either with X-ray and
radium or with X-ray only ; most of the cases were referred for post-operative
treatment.

Four of the 17 cases which were treated with Thio-TEPA were referred prior
to January 1957.

AH cases treated with Thio-TEPA were suffering from recurrence after surgery
and radiotherapy, or the disease was too widespread for effective X-ray treatment
and were inoperable.

The cases were not selected deliberately. AU cases which became known to
one of the authors (0. E.) who were suffering from advanced inoperable carcinoma

treated with X-ray effectively were treated with Thio-
lEPA ; advanced but symptom-free cases were not treated.

It has been our aim to evaluate quantitatively the extent and duration of
objective and subjective improvement.

O. ENGLAJfDER AND A. SARAA^GI

Por approximate quantitative appreciation of the clinical effect ive have
expressed the degree of subjective and objective improvement in five grades and
return to activity has been expressed separately, as shown in the key to the
graphs.

Of the 17 cases treated 3 are alive and 14 died. Six showed no improvement
(Table I), of those six, 3 were in a terminal stage at the start of treatment and had
only two or one injection.

Table l.~Cases in Whom TMo-TEPA had no Effect

Case

First symptom

Duration
from first
sj-mptom
to first

Duration
of ob-
servation
from first

Total dose

Degree of
leukopenia
durine

No.

Age

! and date

treatment

Histology

and route

treatment

Remarks

October 195G.
Enlarged
abdomen
and cough

4 months

24 weeks

Pleomorphic

carcinoma

125 mg.
into tumour

O-

Died 24 weeks after
first injection.

December 1957.
Lassitude

10 months

2 weeks

Adeno-carcinoma

25 mg.
intra-
peritonenl

Died 2 weeks after
first injection.

April 1957.
Swelling of
abdomen

7 months

2 days

Granulosa cell
carcinoma

25 mg.
intra-
peritoneal

—

Died 2 days after
first injection.

August 1957.
Acute abdominal
pain and
vomiting

3 months

2 weeks

Arrhcnoblastoma

50 mg.
intra-
peritonea!

Died within 2 weeks
of injection.

July 1957.
Abdominal
pain

18 months

8 weeks

Anaplastic
carcinoma
? arrhenoblastoma

60 mg.
intra-venous

Died 8 weeks after
first injection.

March 1958.
Cough

3 months

12 weeks

Adeno-carcinoma

100 mg.
intra-
peritoneal

Died 12 weeks after
first injection.

Eleven cases

shoAved a Amrying degree of improA^ement. Eight showed objec-

tive and subjective improvement. Three cases observed for only 11 to 12 weeks
showed subjective improvement only (Table II).

The following case histories, with graphs, illustrate the clinical effect of
Thio-TEPA.

CASE 1 {FIG. 1)

Age 55. Histology : Granulosa cell tumour. First symptom : February
1956, pain in left iliac fossa. July 1956 : removal of bilateral carcinomatous
tumours of ovary. The left ovarian tumour was incompletely removed because
of extensive adhesion ; secondary nodules on peritoneum and in omentum at
the time of operation.

Post-operative X-ray treatment and one radium application to uterus was
given in August/September 1956.

Prior to treatment with Thio-TEPA : severe abdominal pain ; she could
hardly keep any food do^vn because of incessant vomiting. There was a large

EFFECT OF THIO-TEFA OK OVAEIAK TUMOURS

tumour arising from the pelvis extending into abdomen to the level of the
umbilicus.

31.1.57 treatment with Thio-TEPA commenced. She was given a course of
55 mg. in 3 injections into tumour over 3 weeks and later further injections as
shown in graph.

Degree of clinical improvement

Grade 1 . Improvement of symptoms.

2. Disappearance of symptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of symptoms.

Kormal activity

Restricted activity !/ //////A

Within one week from the start of treatment -with Thio-TEPA improvement
commenced, and in the third week vomiting had ceased, pain was markedly
improved and the tumour had become smaller.

In the 9th week she was free from pain. The tumour was greatly reduced
in size and could just be felt above the symphysis. vShe maintained improvement
for 16 weeks, then the symptoms recurred and the tumour grew again rapidly in
Size.

A second course of 4 injections at weekly intervals to a total of 90 mg was
followed by improvement of pain and shght gain in strength for only 6 weeks,

0. ENGLANDER iVND A. SARANGI

Case

No.

Ago

Table II. — Cases in Whom TMo-TEPA

. Time of

Duration from observation

1st symptom to from 1st

I- irst 1st toatraent treatment Total dose

Thm-TEPA tWth Thio-TEPA and route of

and date m months Histology in weeks administration

, 53

February 195G.
Pain in left iliac fossa

Granulosa cell
carcinoma

240 mg. into tumour and
intramuscular

April 105G.
Swelling of lower ab-
domen

Ditto

455 mg. intraperitoneal

March 1058.
Pain in left abdomen

375 mg. intraperitoneal
and intramuscular

May 1958,

Swelling of abdomen

Serous papillary
adenocarcinoma

150 mg. intraperitoneal

December 1955.
Malaise

Granulosa cell
carcinoma

127 mg. intraperitoneal,
intravenous and intra-
muscular

May 1 950.

Pain in right lower
abdomen

Papillary cyst-
adenocarcinoma

112-5 mg. intravenous

October 1958.
Lassitude

Papillary ndeno-
carcinomn

175 mg. intraperitoneal

December 1958.
Swelling of abdomen

No histologj''

120 mg. intraperitoneal

November 1956.
Pain in left pelvis

Granulosa cell
carcinoma

100 mg. intramuscu-
larly

December 1957.
Vaginal bleeding

Poorly differenti-
ated adeno-
carcinoma

215 mg. intraperitoneal
and into tumour

June 1958.
Swelling right groin

Popillary adeno-
carcinoma

200 mg. intraperitoneal

EFFECT OF THIO-TEPA ON OVARIAN TUMOURS

was Effective in Varying Degree

Degree of
leukopenia

during Remarks

Clinical effect treatment June 1 1959

Cessation of pain and vomiting and marked
reduction in size of tumour for 15 ■weeks

+ + -f From 38th ^%’eek onwards
gradual deterioration.
Died 51 weeks after
first injection

Temporary disappearance of tumour ascites -1 — P Still under observation,

and symptoms — returned to normal acti-
vity. Improvement maintained for 32
weeks. Second course of injections for
recurrence followed by disappearance of
ascites, reduction of tumour and improve-
ment of sjTnptoms for 20 weeks. Third
course of treatment started for recurrence,
but no improvement observed jnt

Considerable regression of tumour. Strength,
appetite improved. Greater sense of well-
being

-(- -r Alive and well doing light
housework

Iilarked regression in size of tumour. Ascites -r -h Free from sjTnptoms.
disappeared. All sjmptoms disappeared Enjoying normal life.

— returned to normal activity

Disappearance of oedema of legs. Disten- -1-
sion of veins of abdominal wall diminish-
ed. Improvement of nausea, vomiting
and pain. Slight gain in appetite and
weight

Retrogression of pelvic tumour. Pain de- -}- -f -f
creased. Appetite and strength im-
proved

Developed recto-vaginal
fistula in the 11th week.
U nder consideration for
a further course when
white count improves
Improvement main-
tained.

Ascites decreased. Gained strength. Ap-
petite improved. Sense of well-being

Doing her housework and
working as part-time
tj-pist

Appetite and strength improved

Symptoms improving

No measurable regression of tumour.
Gained strength

Improvement of sjmp-
toms maintained to
date

Pain and ascites diminished. Sense of well- -f- Died 16 weeks after first
being started within two weeks following injection of Thio-TEPA

first injection. Improvement maintained
for 12 weeks. Slight reduction of tumour
noted

Improvement in strength and appetite only -i- -f -f Died 12 weeks after first
for a short period injectionofThio-TEPA

Further observations
December 1959

Patient died 19. viii. 59 ; 92
weeks after the first in-
jection of Thio-TEPA.

Patient died 8.x. 59; 47

weeks after the first in-
jection of Thio-TEPA.

Well until August 1959 then
recurrence of growth and
ascites. Further injec-
tions of Thio-TEPA fol-
lowed by disappearance
of sjmptoms for 8 weeks.
Then rapid progress of
disease and failing
strength.

Patient died 9.vi.59; 19
weeks after the first in-
jection of Thio-TEPA.

Improvement maintained.
Two intramuscular in-
jections of 30 mg. each
were given in December.

Improvement maintained.
Further 140 mg. in 6
intra - peritoneal injec-
tions given.

Patient died 20. vii. 59 ; 18
weeks after the first in-
jection of Thio-TEPA.

Patient died 4.xi.59; 30
weeks after the first in-
jection of Thio-TEPA.

0. ENGLANDBE AND a. SARANG]

injection“^’^““" &st

T'® f^f leucocytes was of particular interest. The number of leucocytes
at the start of treatmei^ was 3000, dropped to 1700 in the 9th week after\he

commlZlT ^ quickly rose to 7100 when clinical improvement

Time of observation was 51 -weeks.

CASE 2 (fig. 2)

Age 50. Histology : well differentiated, probably granulosa cell carcinoma
First symptom ; April 1956, swelluig of lower abdomen.

Fig. 2

Degree of clinical improvement

Grade 1. Improvement of sj’mptoms.

2. Disappearance of sjmiptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumom-, of ascites and of sjTnptoros.

Normal activity
Restricted activity

13.8.56 subtotal hysterectomy and bilateral oophorectomy for bilateral
ovarian carcinoma. Secondary nodules on pelvic peritoneum and peritoneum
overlying the bladder. 13.8.56 2 pints of blood transfused.

September/October 1956 post-operative X-ray treatment and one radium
application to uterus.

Prior to Tliio-TEPA : pelvic pain, abdominal distension and bleeding from
the umbilicus on slight trauma. Examination after removal of 3000 ml. of ascites
by paracentesis on 12.11.57: nodular mass in pouch of Douglas extending into
both sides of pelvis. Secondary tumour, 2 cm. diameter, in umbilicus.

EFFECT OF THIO-TEPA ON OVAEIAN TUMOURS

Commencing on 13tli November 1957, she was given the first course of Thio-
TEPA, 3 intra-peritoneal injections of 25 mg. each, in 3 weeks. She vomited
occasionally during the first 7 days ; from the second week onwards she gained
strength, her appetite improved and she felt well. From the 8th week onwards
there was no evidence of disease and she was free from symptoms. Definite
decrease in size of the tumour was observed in the 7th week after the first injection
and in the 1 1th week all gro-wth and ascites had disappeared. She led a normal
life doing all her housework for approximately 8 months, then pain, ascites,
tumour of pelvis and umbilicus reappeared rapidly.

She was given further injections of Thio-TEPA commencing Avith a course of
4 injections of 25 mg. each in 4 weeks, and later further injections as sho-wn in
Fig. 2.

Six weeks after the start of the second course she again gained in strength,
attained a sense of well-being and her appetite improved ; her symptoms disap-
peared completely and she returned to her normal activities for 3| months.
During this period ascites disappeared, the grovdh had decreased in size but did
not disappear ; then pain and ascites recurred rapidly and frequent paracentesis
became necessary. Further injections were given as shown in the graph.

The patient died 19.8.59, 92 weeks after the first injection of Thio-TEPA.

CASE 3 (fig. 3)

Age 44. Histology : granulosa cell carcinoma producing pseudoadenomatous
appearances.

First symptoms ; March 1958, pain in left abdomen.

Operation; 24.9.58; gross ascites, abdomen filled -with bilateral multi-
locular ovarian cystic tumours with a large amount of solid and friable tissue.
Metastases on pelvic peritoneum and uterus. Both ovarian new growths removed.

Prior to Thio-TEPA ; 27.10.58 ascites ; firm mass of growth in upper third of
rectovaginal septum and pouch of Douglas ; large mass in left pelvis and a large
mass in left lumbar region extending into left hypochondrium.

Treatment with Thio-TEPA commenced on 10.11.58 ; a course of 84 mg.
was given in 5 injections in 10 days, and a second course of 116 mg. commenced 8
weeks after the first injection.

From the 10th day onwards she gained strength, her appetite improved and
later on she felt well and was doing light housework except when ascites had
accumulated. From the 14th week onwards tumour decreased in size but did not
disappear. In the 19th week a paracentesis of abdomen was necessary,
subsequently she was well and doing light housework until the 25th week when
ascites and symptoms recurred. Further injections were given as shown in
Fig. 3.

The patient died 8.10.59, 47 weeks after the first injection of Thio-TEPA.

CASE 4 (fig. 4)

Age 52. Histology ; serous papillary adenocarcinoma of ovary
First symptom ; May 1958, swelling of abdomen.

0. ENGLANDER AND A. SARAN6I

Operation 13.8.58: 4500 ml. of ascitic fluid removed
turner ona diameter ,e„.„ved. PeritenSiee^S m
13.8.58: 4 pints of blood transfused.

uvaiiaiLl

Fig. 3

Degree of clinical improvement

Grade I. Improvement of symptoms.

2. Disappearance of sjTnptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of symptoms.

Normal activity

Restricted activity /////'. ' ^ / A

Post-operative X-ray treatment September/October 1958, could not be
completed because of poor general condition.

Prior to Thio-TEPA : November 1958, examination under anaesthesia after
removal of 5700 ml. of ascites : a mass consisting of matted spherical tumours
filling pelvis and lower abdomen extending into upper abdomen on the right side.

Main symptoms : lassitude and gnawing abdominal pain.

EFFECT OF THIO-TEPA OK OVAKIAK TTOIOUBS ^ '

On 22 . 1 1 . 58 the first course of Thio-TEPA commenced ; 100 mg. Avere given
in 4 intra-peritoneal injections of 25 mg. each at weekly intervals, and she was
given a further 25 mg. in the 16th and 17th week after the first injection.

In the 6th week decrease in size of tumour mass was observed, and in the 10th
week the tumonrs had disappeared except for one 4 cm. diameter lump at the

Fio. 4

Degree of clinical improvement

Grade 1. Improvement of symptoms.

2. Disappearance of symptoms.

3. Decrease in size of tumom or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of symptoms.

Normal activity
Restricted activity

level of umbilicus which was still palpable. At that time ascites had disappeared
and did not re-acoumulate during the time of observation. The lump at the level
of umbihcus continued to decrease in size but did not completely disappear.
From the second week onwards her appetite improved, she gained strength and
attained a sense of well-being, and from the 18th week onwards she could do all
her housework, and was enjoying a normal life.

She remained well and vigorous and the tumours decreased further in size
until August 1959. In August the tumours, ascites and symptoms reappeared.

O. ENGLANDER AND A. SARANGI

bhe g.o,Hh sr::xrcfs

CASE 5 (pig. 5)

Age 55. Histology : granulosa cell carcinoma.
iDrst symptom : December 1955, malaise.

. .1.56. removal of a large left multilocnlar C 3 ^stic ovarian grovlili.

JAN FEB MAR. APR. MAY TUME

iqs'i

Fig. 5

Degree of clinical improvement

Grade 1. Improvement of sjTnptoms.

2. Disappearance of sjTnptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of sjTnptoms.

Normal activitj'
Restricted activitj^

ty/yyyA

f/> // A // / A

Maj'^ 1957 : large fixed hard nodular mass of growth in pouch of Douglas and
left pelvis. X-ray treatment and one radium application to uterus was followed
by only slight regression of grovdh and temporary alleviation of pain lasting
approximately 6 months.

Prior to treatment with Thio-TEPA : January 1959, extensive growth involv-
ing rectum and rectovaginal wall causing stenosis of rectum, oedema of legs.

EFFECT OF THIO-TEPA ON OVARIAN TUJIOURS

vulva and buttocks and gross distension of veins of abdomen. She had nausea,
occasional vomiting, anorexia, frequent watery stools and sometimes loss of
control, frequencj'^ of micturition, abdominal pain and tiredness even at rest :
she was still doing a little light housework.

Treatment mth Thio-TEPA started on 27.1. 59. She was given a course of
6 injections to a total of 100 mg. in 3 weeks and then one 27 mg. intra-muscularly
9 weeks after the first injection.

Prom the second week onwards pain and nausea decreased, appetite improved
and she gained a little in strength.

The tumour decreased in size slightly and the oedema of legs, vulva, buttocks
and the distension of abdominal veins disappeared. A rectovaginal fistual
developed in the 12th week. The distension of abdominal veins and oedema of
thighs and pain reappeared in the 14th week.

A colostomj'^ was done and one pint of blood was transfused on 4.6.59.

The patient died 9.6.59; 19 weeks after the first injection of Thio-TEPA.

CASE 6 (fig. 6)

Age 54. Histology ; papilliferous cystadenocarcinoma.

First symptom : May 1956, pain in right lower abdomen.

15.9.58: complete removal of left ovarian growth, incomplete removal of
large friable growth of right ovary which was adherent to uterus and pelvic wall.

17.9.58; 2 pints of blood transfused.

October /December 1958 : one radium application to uterus and X-ray
treatment to pelvis.

Prior to treatment with Thio-TEPA ; a large mass in the pelvis involving
uterus pressing on rectum. Constant pain in abdomen and in right shoulder for
which she required alternatively papaveretum gr. ^ or pethidine 100 mg. 4 hourly
day and night. She was very ill and confined to bed.

5.12.58; 2 pints of blood transfused.

Treatment with Thio-TEPA commenced on 10.2.59.

She was given at first 62-5 mg. in 7 intra-venous injections in 17 days.

From the 6th week onwards her appetite improved, she gained strength and
pain decreased. Decrease in size of the tumour commenced in the 8th week ;
in the 9th week abdominal pain had almost completely disappeared. She was
discharged home and in the 12th week she could walk around and help a little in
her household.

A further 2 intra-muscular injections of 30 mg. of Thio-TEPA were given in
December. Objective and subjective improvement was maintained.

CASE 7 (fig. 7)

Age 54. Histologj”^ : papillary carcinoma of ovary.

First symptom : October 1958, lassitude.

12.11.58 operations ; gross ascites. Omentum and peritoneum studded with
nodu es, pehns .Sljed irith groui:h. During the 2 months proceeding the first

Tlno-TEPA she needed paracentesis on 6 occasions at intervals of
0 to 21 days.

0. ENGLANDER AND A. SAEANGI

Prior to Tiiio-TEPA : gross distension of abdomen due to large masses of

Thio-TEPA commenced. She was given as the
first course 150 mg. divided into 5 mtra-peritoneal injections at weekly intervals
further injections as shown in Eig. 7.

Fig. G

Degree of clinical improvement

Grade 1. Improvement of sjmptoms.

2. Disappearance of sjmiptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of symptoms.

Normal activity
Bestrieted activity

r/ // / /■ /

During the first 2 weeks after the first injection a paracentesis was done on
two occasions. From the 3rd week onwards ascites decreased slightly. She
gained strength, her appetite improved and she felt well.

She is doing her shopping and working part time as a typist.

Change in the size of the tumour is difficult to assess because of ascites.

The time of observation since the first injection of Thio-TBPA was 15 weeks
on June 1st 1959.

EPFECT OF THIO-TEPA ON OVARIAN TUMOURS 41

From June 1st till October 9th 1959 she was given a further 140 mg. in 6
intra-peritoneal injections. Paracentesis abdominis was necessary once in
October 1959.

Degree of clinical improvement

Grade 1. Improvement of symptoms.

2. Disappearance of symptoms.

3. Decrease in size of tumour or decrease of ascites.

4. Clinical disappearance of ascites.

5. Disappearance of tumour, of ascites and of sjTnptoms.

Normal activity

Restricted activity !//////■ / X

At the end of December ascites and abdominal tumour were present ; she
felt quite well except at the time when a paracentesis had become necessary ;
she continues doing part time work as a typist and also doing light housework.

CASE 8

Age 55. No histology.

Pirst symptom : December 1958, swelling of abdomen.

Prior to treatment with Thio-TEPA ; distension of abdomen, shortness of

O. ENGLANDER AND A. SARANGI

breath loss of weiglit and anorexia. A paracentesis of chest and abdomen was
done 5 times during the preceding 2 months.

Examination after removal of 7 litre of fluid by paracentesis of abdomen •
large nodular grovdh m pelvis fillmg pouch of Douglas extending upwards to
the level of umbilicus. Multiple large lobulated tumour masses in abdomen
Liver enlarged.

Treatment with Thio-TEPA commenced on 18.3.59. She was given 4
intra-peritoneal injections at weeldy intenmJs to a total of 120 mg. Ascites was
removed by paracentesis on 4 occasions during the 6 weeks following the first
injection. From the 5th week onwards she gained strength and her appetite
improved.

In the Otli week ascites and pleural effusion mcreased causing dyspnoea and
therefore fluid was removed by paracentesis of chest.

The patient died on 20.7.59, 18 weeks after the first injection of Thio-TEPA.

CASE 9

Age 45. Histolog}' : granulosa cell carcinoma.

First symptom ; November 1956, pain in left pelvis.

March 1957 ; h 3 ^sterectomy and bilateral salpingo-oophorectom}^ for carcinoma
of ovary.

April /Maj' 1957 : post-operative X-ray treatment to pelvis.

Juty 1958, secondary'- node in operation scar treated vith X-ray.

klass of growth in pelvis treated with X-ray November /December 1958.

Prior to treatment with Tliio-TEPA ; a fixed hard mass in left pelvis extending
almost to the level of umbilicus. 4 cm. diameter subcutaneous metastatic tumour
in abdominal v'all in upper end of operation scar. Liver enlarged. Slight pain
in right iliac fossa, slight backache and nausea.

Treatment ndth Tliio-TEPA commenced on 18.3.59. She was given at first
3 injections intra-muscularlj'^ of 25 mg. each at weekly intervals.

The leucocytes dropped from 6400 to 2300 in the 3rd week ; a further 25
mg. was given after the leucocjdes had risen to 4200 at the end of the 6th week.

From the second week onwards she gained strength and attained a sense of
well-being. No definite change in the size of the tumour has been observed.

The patient died 4.11.59, 30 weeks after the first injection of Thio-TEPA.

METHOD

The crjrstalline Thio-TEPA was dissolved in normal saline 10 mg. per nil. and
stored no longer than 14 daj^s in refrigeration. The route and dose in this series
varied. If ascites was present the injections were given into the peritoneal camty.
A small quantity of 2 per cent procaine was injected locally before giving Tluo-
TEPA intra-muscularly or into the tumour. The dose varied from c^e to case
depending on the patients strength, the state of nutrition, haematological status
and on the route. From our experience we came to the conclusion that 6 o
120 mg. given in fractionated doses over tliree to four weeks is an eflec lye
therapeutic dose. Intravenously Thio-TEPA was given at the rate ^ °

mg. per day to a weeklj^ maximum dose of 30 mg. Injections of Thio-IL ar
discontinued when the graph of total leucocytes showed a downward trena
following previous injections.

EFrECT OE THIO-TEPA ON OVARIAN TUMOURS

Principles of maintenance treatment are yet to be developed. We intend to
give a second course in all cases which show a good response, at the latest 3 to 5
months after the first course.

It is of great value that most cases can he treated as out patients.

EPFECT AND REACTIONS

In 4 of our cases of advanced carcinoma of the ovary, treatment with Thio-
TEPA was followed by strildng temporary regression or clinical disappearance of
the grovdh, reduction or disappearance of ascites and marked improvement of
symptoms with return to activity and enjoyment of life. Three patients had
definite marked temporary regression of growth and improvement of symp-
toms only. The maximum duration of improvement after one course was 8
months. Second and subsequent courses were less effective than the first course.

Improvement in strength, appetite and sense of well-being, soon after the
start of treatment, was noted in all cases in whom Thio-TEPA had any clinical
effect.

Though symptoms may improve during or shortly after the second week,
decrease in size of tumour and of ascites occurs mostly after 6 to 8 weeks.

All our cases who showed a marked improvement had temporary leukopenia
following treatment.

Decrease in the number of granulocytes and platelets commenced in the
second or third week, and recovered quickly to normal. Petechiae and cutaneous
haemorrhages were observed in two cases at the time of maximum depression of
haemopoiesis and subsided spontaneously.

The effect of Thio-TEPA on leucocytes was remarkable in case 1 in whom the
number of leucocytes at the start of treatment was 3000, dropped to 1700 8 weeks
after treatment and rose to 7100 one week later. The fall and later the rise of
the number of leucocytes coincided with retrogression of growth and improvement
uf symptoms.

Only one case had side reactions such as nausea and vomiting. There were
no unpleasant local or general reactions in any of the other cases.

DISCUSSION

Thio-TEPA has a marked growth inhibiting effect in some cases of carcinoma
of the ovary. The effect is only temporary. Thio-TEPA does not supersede
surgery or radiotherapy in the treatment of ovarian cancer. It is, however, of
definite value in some cases in whom all other treatments have failed. In such
cases Thio-TEPA may still achieve temporary control of disease to such an extent
that the patient may enjoy a normal life and may return to normal activity for
many months. The duration of temporary control will, one can reasonably
expect, increase with greater experience and by the various means of protecting
the haemopoietic tissue. °

Small repeated transfusions of blood given daily for a few days before and
during treatment vuth Thio-TEPA, may reduce the proliferative and mitotic
activity of haemopoietic tissue to a resting phase. In the resting phase the
bone marrow may be less sensitive to a radio-mimetic and antimitotic agent

If this concept is correct, 50 ml. of blood given daily should reduce the bone
marrow activity. A plan for investigation in this direction is in preparation

O. ENGLANDER AND A. SARAE'GI

Prom w]iat has already been done in this field, and from our own experience
we conclude that ovarian carcinoma occupies the foremost position in the spectrum
of activity of Thio-TEPA over the whole range of malignant disease.

In many cases of advanced cancer classification by histology is often difBcult
and sometimes debatable. It is likely that some cases wliich appear to be primary
malignant tumours may, in fact, be secondaries from a growth elsewhere. H
this is true the selective effect of Tliio-TEPA on primary ovarian carcinoma
is probably higher than it appears from our series. It is of immediate interest
to correlate sensitivity of the various groups of ovarian cancer to chemotherapeutic
agents wth their natural history and histology, and to study each group according
to their sensitivity,

A plan of study of the effectiveness of chemotherapy as an adjuvant to surgical
treatment of cancer has been described by Shimkin and Moore (1958).

Two investigations are being carried out using Thio-TEPA in resectable
carcinoma of stomach and nitrogen mustard in resectable cancer of lung. One of
the aims of the plan is to ascertain whether chemotherapeutic agents are more
effective when the tumours are small and clinically not obviously established.

The discover3’' of the effect of Thio-TEPA on carcinoma of ovary has opened
new and promising paths in the field of experimental and clinical research on
ovarian carcinoma.

straniARy

The effect of Thio-TEPA in 17 cases of advanced carcinoma has been described.

Objective improvement has been achieved in 8 cases, some of whom have
returned to their normal actimty for several months. The clinical effect is
temporary. In all cases the effect was associated with transient leukopenia.

We are most grateful to the Lederle Laboratories, Division of Cyanamid of
Great Britain Limited, for their generous supply of Thio-TEPA and for literature
Q'Tid rofcrciiccs.

We thanli hlrs. Sheila Cocks, M.S.R., Superintendent Radiographer, and the
Photographer, Mr. Brooks, for the reproduction of the graphs.

We also thank Miss Pam Martin for her secretarial help.

REFERENCES

Alpert, L. K. — (1958} Ann. N.Y. Acad. Sci., 68, 107^ , r , oco c7q
Bateman, J. 0.-(1958) Ibid., 68, 1057.-(1955) Nm Engl. J. Med., 252, 879
Leonabd, B. J., Israels, M. C. G. and Wilkinson, J.— (1956) Lancet, u, 1017.
Leone, L. A.— (1958) Ann. N.Y. Acad. Sci., 68, 1081.

Moore, G. E. — (1958) Ibid., 68, 1074.

Olson, K. B.— (1958) /6fd., 68, 1018. t o n w Arch

Shay, H., Laeaeonetis, C., Sjoth, N., Weldow, I. and Sun, C. H.— (1953) Arch.

intern. Med., 92, 628. , n • nn mAc

Idem AND Sun, C. H.— (1958) Ann. N.T. Acad Sen, 68, 1046.

Shimkin, M. B. and Moore, G. E.-(1958) /. Amer Acad Sci 68

Ultman, j. E., Hayman, G. A. and Gellhorn, A.— (1958) Ann. A .1 . Acad, c ., ,

1007. „ . , T • 101K

Watson G. W. and Turner, R. L. — (1959) Bnt. vied. J., i, lylu. . , „ . on

c., Golomb, P. M. and Gu^iport, S. L.-(1958) Ann. N.7. Acad. Sci., 68,

937 .

THE ISOLATION OE EPITHELIAL CELLS FROM
NORRIAL AND NEOPLASTIC COLON

R. A. DALE

Fro 7 n the Department of Chemical Palhologjj, Postgraduate Medical School of London,

W. 12

Eeceived for publication December II, 1059

The comparison of the enzyme activities of normal and neoplastic tissue is
usually unsatisfactory because it is carried out on blocks of tissue containing a
mixed population of cells. The ratio of the epithelial to the stromal comjponent
varies not only as between normal and neoplastic tissue, but also within different
normal and neoplastic tissues. Attempts have been made to resolve this difficulty
by counting the relative numbers of each kind of cell (Chalkley, 1943 ; Rosenthal
and Drabkin, 1944 ; Sibley and Fleisher, 1955). However, tliis is a tedious pro-
cedure and it does not overcome the problem of determining the amount of
enzyme activity in each different component of the tissue.

Clearly, the epithelial component of both the tumour and the homologous
normal tissue should be isolated in order to compare their enzyme activities. In
this paper such a procedure is described for the isolation of the epithelial cells of
the mucosa and of carcinomata of the colon.

METHODS AND BESDLTS

Preparation of tissue

The colon is obtained within 30 minutes of excision. A longitudinal incision is
made along its whole length, the mucosa is wiped free from faecal matter with the
aid of filter paper and the subperitoneal fat is removed. The specimen is then
pinned out on a board and the mucosa is separated from the submucosa at or
above the level of the muscularis mucosae by means of a stiff paint or tooth brush
the bristles of which have been cut to 0-5 cm. in length. The sheets of mucosa
obtained in this way are transferred to ice-cold 0-25 m sucrose.

The everted edge of the tumour is removed above the levels of the adjacent
normal mucosa externally and the ulcer base internally. The tissue so obtained
is compressed between sheets of filter-paper in order to remove mucus and necrotic
debris and it is then trimmed to remove the connective tissue and any areas of
haemorrhage.

The pieces of normal mucosa and tumour are finally washed three times in ice-
cold 0-25 M sucrose. At this stage the histological appearance is that seen in Fig. 1,
2 and 3. In Fig. 1 a section of the mucosa superficial to the muscularis mucosae is
seen, and in Fig. 2 there is a section of the growing edge of a carcinoma of the
colon. In both of these sections the epithelial cells and stroma are clearly defined
from each other. In Fig. 3 there is a surface view of a piece of fresh mucosa mounted
under a coverslip in 0-25 m sucrose. The acini and the individual ceUs of which
they are compnsed are seen in plan in situ in the lamina propria When the

R. A. DALE

microscope is focussed up and down on such a preparation the lumina of the acini
have tJie appearance of tunnels.

Isolation of epithelial cells

The epithelial cells are now isolated by a combination of two processes, namely

(1) Disruption of the tissue, and

(2) Repeated differential centrifugation of the resultant suspension. The
details are set out below.

All materials and equipment are kept at 0° C. and all procedures except weigh-
ing are carried out at this temperature. A histological examination is carried out
as follows on each sample of the suspension removed for high-speed centrifugation.
The sample is removed from the centrifuge tube by means of a Pasteur pipette
which is filled from the surface of the suspension. This ensures that the deeper
la 3 mrs, i.e., the last to be removed, are situated in the distal part of the pipette.
At stages 1(c), 2(b) and the corresponding stage under 3 of the scheme below,
one drop from the tip of the pipette is placed on a glass slide, stained and examined
for the presence of stroma. If anj’^ stroma is seen a suitable volume of the sus-
pension is returned to the centrifuge tube, and again the drop in the tip of the
pipette is examined. This procedure is repeated until aU of the stroma is elimin-
ated. The remainder of the contents of the pipette is then discharged into tube 0
for high-speed centrifugation. After a little experience onlj’^ 3-4 histological
examinations are needed.

Scheme for the isolation of epithelial cells from stroma

1. (a) 1 g. tissue plus O-O ml. 0-25 m sucrose are placed in the tube of a Potter-
Elvehjem t 3 'pe homogeniser (tube A), and the pestle* is forced down and up 20
times.

(b) The suspension is centrifuged (in tube A) at 2500 r.p.m. for 3 minutes and
the supernatant liquid plus the upper half of the “ flufF 3 ' ” la 3 mr (see later) are
removed to a second tube, B.

(c) Tube B is centrifuged at 2500 r.p.m. for 3 minutes and the upper three
quarters of the suspension are removed to tube C.

(d) Tube C is centrifuged at 5000 r.p.m. for 5 minutes, and the clear super-
natant liquid is transfered to tube B, mixed with the contents remaining as under
(c) and poured into the homogeniser.

2. (a) The pestle is forced down and up a further 80 times and the contents
of the homogeniser are returned to tube B.

(b) Tube B is centrifuged at 2500 r.p.m. for 3 minutes and the upper half of
the suspension is transferred to tube C.

(c) Tube C is centrifuged at 5000 r.p.m. for 5 minutes and the clear super-
natant liquid is transferred to tube B which is swirled in order to mix the contents.

3. The procedures 2(b) and (c) are repeated until the number of free nuclei
remaining in the deposit in tube B is negligible, that is, approximate^ 5 per
high-poM^er field. Usually it is necessary to repeat procedures 2(b) and (c) twice

more.

* A plastic pestle is used. The clearance between the wall of the tube and
such that when the pestle is held vertically with the tube m position and containing uater, the

tube slowly falls.

ISOLATION OP EPITHELIAL CELLS EROIM COLON

Disruption of the tissue

The movement of the pestle against the homogeiiiser tube compresses the
tissue in such a way that the epithelial cells are expressed from, and/or stripped off,
the stromal tissue. The appearance in the fresh state of the suspension so produced
from the mucosa is seen in Pig. 4, 5 and 6. In Pig. 4 the expression of the acini
from the lamina propria is shovm at an early stage and in Pig. 5 an acinus is shown
lying free surrounded by the nuclei of disrupted cells. The appearance of the
lamina propria is seen in Pig. 6 in wliich the complete removal of the acini is
demonstrated. A section of the tumour in the same stage of preparation appears
in Pig. 7 in Avliich clumps of tumour cells and fragments of connective tissue are
visible.

Differential centrifugation

As a result of centrifugation the suspension is resolved into the follomng
layers from below upwards : pieces of connective tissue containing a few acini,
clumps of cells, single cells, nuclei, mitochondria and microsomes. The connective
tissue, cells and nuclei are usuallj^ held together as a pink fluffy layer by the mucus
which is released from the goblet cells. This makes it difficult to separate the
various components of the suspension. However, separation is acliieved, as out-
lined above, by a system of centrifugations followed by washing of the deposit
with the top layers of the supernatant liquid. The end-result is apparently a
complete resolution of most of the epithelial cells from the stroma.

The appearance of sections of the epithelial cells and nuclei prepared in tliis
way from the mucosa are seen in Pig. 8 and 9. Many of the acini and separate
cells appear to be intact and even -where the ceU membrane was broken the nuclei
do not seem to have been damaged. Most of the cells and nuclei are clearly epi-
thelial, but there is a small percentage of nuclei whose origin it is impossible to
ascertain. It is unlikely that many of these nuclei arise from the connective tissue
because, as sho^vn in Pig. 10, the lamina propria does not appear to have been
disturbed by the procedure used to express the acini.

The same general comments apply to the sections prepared from the epithehal
cells and connective tissue residue of the carcinoma. In Pig. 1 1 both the malignant
cells and the free nuclei appear to be intact. In Pig. 12 the cotmective tissue of
the tumour is still cellular but owing to the lack of regular architecture it is
difficult to be certain that none of the cells ffom the connective tissue was shorn
off in the homogeniser.

DISCUSSION

This appears to be the first occasion on wliich epithelial cells have been
isolated from the supporting elements in human tissue on a large scale. Hele
(1953) prepared epithelial cells from the small intestine of the rat using a similar
technique. ®

There are at least three criticisms of the method. First, the epithelial cells may
be contaminated with connective tissue cells. As already pointed out this does
not seem to be a likely or important source of error. Second, some of the contents
ot the cells of the stroma may leak into the sucrose medium. At present there is
0 ° O whether this has happened, but it is probably minimal at

0 0. Third, all of the epithehal ceUs are not recovered. Complete recovery was

B- A. DALE

not attempted because it is believed that the less the disruntion nf fv
commensurate with an adequate yield of epithelial cells, the less is the itelihood
tLsrcSs.'''^ "" comiective tissue and of leakage of enzymes from

There appears to be no reason why the method should not be applied to other
tissues, for example to small intestine in man and to tumours in which the epi-
thelial cells are readily removed from the stroma in man or animals Several
attempts were made to obtain a preparation of epithelial cells in this way from
human gastric mucosa ; tliej' failed because in the stomach the acini ^ not
separate readily from the lamina propria.

SUaiMAKY

A method for the isolation of the epithelial cells Srom human colonic mucosa
and carcinoma is described.

My thanks are due to Dr. Basil Morson and the staff of the Research Depart-
ment at St. Mark’s Hospital and to surgical colleagues at several hospitals for the
supply of tissue, to Dr. I. Doniach for his admce and criticism and to Jlr. W.
Brackenburj’’ for the photomicrographs.

The author is a Saltwell Fellow of the Rojml College of Physicians and is also
in receipt of a grant from the British Empire Cancer Campaign.

REFERENCES

Chalklev, H. W.— (1943) J. iia/. Cancer Inst., 4, 47.

Hele, M. P.— (1953) BiocJiem. J. 55, 857.

Rosenthal, 0. and Dbabkin, D. L.~(1944) Cancer Bes., 4, 487.

Sibley, J. A. and Fleishee, G. A.— (1955) Ibid., 15, 609.

EXPLANATION OF PLATES

Pig. 1. — Section of the mucosn of the colon prepared from the sheets of mucosa stripped off the
submueosa with the aid of a stiff brush. X 60,

Fig. 2. — Section of tlie growing edge of « carcinoma of the colon shoiving the tissue from which
the epithelial cells of the carcinoma are isolated. X 60.

Fig. 3. — Surface vie\v of the mucosa showing the acini in plan (unfixed). X 57.

Fig. 4. — Expression of the acini from the lamina propria at an early stage (unfixed). X 57.

Fig. 5. — Extruded acinus lying among the intact nuclei of epithelial cells (unfixed). X 80.

Fig. 6. — Surface view of the lamina propria from which the acini were expressed (unfixed).
X 5 / .

Fig. 7. — Clumps of tumour cells and connective tissue as seen during disruption of a carcmoma
(unfixed), x 60. , . • i *

Fig. 8. — Section of the epithelial cells and nuclei isolated from the lamma propria. Note tnat
many of the acini are intact, x 60. , . cnn

Fig. 9. — High-power view of Fig. 8 showing that the cells are apparently mtact. x bye.

Fig. 10. — Section of the lamina propria showing that the connective tissue cells are still m situ.

X 58.

Fig. 11. — Section of some epithelial cells of a carcinoma isolated from the strorna. X 180.

Fig. 12.— Stroma of a carcinoma after isolation of the epithelial cells shomng that the con-
nective tissue cells are still in situ. X 60.

British Journal or Canxer.

Vol. XIV, No. 1

Dale.

BniTisH .Tootxai, of CAxrEn-

Vol. XIV No. 1

Dale.

BASOPHIL ADENOIMATA IN THE RAT HYPOPHYSIS
AFTER GONADBCTOMY

W. E. GRIESBACH and H. D. PURVES

From the Endocrinology Research Department, Netv Zealand Medical Research Council,
Medical School, Dunlin, Neiu Zealand

Received for publication Januarj'^ 28, 1960

The production of neoplasia by prolonged stimulation of endocrine tissues by
physiological mechanisms is not only of great intrinsic interest but has some
clinical significance because of the probability that such mechanisms are operative
in the production of neoplasms in man. An analogy has been draAvn by Purves
(1956) between the production of basophil adenomata in the rat hypophysis by
conditions that cause hyalinisation of normal basoplril cells, and the association
of basophil adenomata with Crooke’s cell changes in the human hypophysis.

The basophil cells of the pars anterior of the rat hypophysis are composed of
two functionally distinct groups. One group secretes th 3 Totrophin and is strongly
and specifically stimulated by thyroxine deficiency. The other group secretes
gonadotrophins and is strongly and specifically stimulated by gonadal hormone
deficiency. The two groups have been termed thjrrotrophs and gonadotrophs
respectively (Purves and Griesbach, 1951). Thyrotrophs and gonadotrophs can
be differentiated by their responses to staining by certain dyes. After staining
by aldehyde-fuchsin the thyrotrophs appear as beta cells with positively stained
granules, while the gonadotrophs appear as delta cells since their granules are
not stainable by aldehyde-fuchsin (Halmi, 1950). There is evidence that the
gonadotrophic group is heterogeneous and comprises two specific types — ^FSH
and LH cells — which secrete folhcle stimulating and luteinising hormone respec-
tively (Purves and Griesbach, 1954, 1955).

Chronic stimulation of the thyrotrophs of the rat pars anterior by thyroxine
deficiency induced by goitrogen administration gives rise to basophil adenomata.
These adenomata are beta cell adenomata composed of angular cells resembling
normal th 3 n’otrophs and containing granules wliich are stainable by aldehyde
fuchsin. Evidence for the view that such adenomata are derived from thyrotrophs
has been summarised by Bielschowsky (1955). It is notable that the cells of the
adenomata do not show any of the hyalinisation changes that affect the majority
of the thiuotrophs in the surrounding pars anterior tissue in rats receiving
goitrogen (Purves, 1956).

Basophil adenomata appearing in the hypophyses of rats after gonadectomy
have been reported by Houssay, Houssay, Cardeza and Pinto (1955) and by
Griesbach and Purves (1956). These adenomata were composed of cells which
resembled normal gonadotrophs but did not show the hyahnisation which affects
the normal gonadotroph after gonadectomy. Specific granulation was often
present in amounts conferring strong staining properties on the adenomata
The granulation was of the delta type since, like that of normal gonadotrophs,
4

W. B. GEIESBACH AND H. D. PUEVES

it was not stained by aldebj-^de-fuchsin (Griesbach and Purves, 1956 ; Purves
1956). Houssay et al. (1955) considered that oestrogen or androgen secreted by
adrenalcortical tumours was involved in the production of the hj-pophysial
adenomata which appeared in tlieir gonadectomised rats. Tliis ^dew was in
accordance vdth the mechanism proposed by Woollej’^ and Dickie (1949) for the
production of hirpopbysial adenomata in castrated mice. We, at one time
considered that gonadectomy at an early age might be necessary for the production
of basophil adenomata since we had not observed such adenomata in an earher
study of the long term effects on the hj^^oplij^sis of rats of gonadectomy performed
on sexually mature rats. iS^either of these h 3 y)otheses is sustained by the
observations reported in this paper.

JUTERLVLS AND METHODS

Both male and female rats of our Wistar strain were gonadectomised in 1953
and 1954 in connection vdth another investigation. The long term survivors
were examined for the presence of h 3 'poph 3 'sial tumours. Some of these rats
had been gonadectomised at 6 weeks of age, (Group A), some at 3 months, (Group
B), and some at 9 months, (Group C).

In addition to the above animals, an experiment was begun m 1954 to determine
the effect of exogenous oestrogen on the response of the h 3 'poph 3 'sis to gonadec-
tom 3 '. This experiment included :

Group D. 24 rats (12 male, 12 female) gonadectomised at 6 weeks
of age and simultaneously implanted with 10 mg. cholesterol pellets
containing 2-5 per cent stilboestrol.

Group E. 24 rats (12 male, 12 female) treated similarty to Group D,
but with cholesterol pellets containing 0-5 per cent of stilboestrol.

Group E. 30 females implanted at 6 weeks of age with 10 mg,
cholesterol pellets containing 2-5 per cent stilboestrol.

Group G. 26 female rats implanted at 6 weeks of age with 10 mg.
cholesterol pellets containing 0-5 per cent stilboestrol.

In 1956 an experiment u'as begun to determine the relation of the adrenal
cortex to hypoph 3 "siaI adenoma formation. This experiment comprised :

Group H. 60 rats (30 male, 30 female) gonadectomised at 6 weeks
of age.

Group I. 60 rats (30 male, 30 female) gonadectomised and adrena-
lectomised at 6-7 weeks of age.

Group J. 38 female rats adrenalectomised at 6-7 weeks of age.

The adrenalectomised animals were given 1 per cent saline as druiking-water.
In some groups, one half of the animals were lolled at IS or 19 monBis after
gonadectomy and the remainder at 24 months. In Groups D, E, F, G, some
animals were killed at earher ages to determine the time of appearance of tumours.

Animals which appeared to be unhealthy or obviousl5^ sick were lolled an
examined grossly. Their h 3 ^ophyses were not examined microscopicalJj'.
The healthy animals were lolled udth coal gas and the h 3 Tophyses removed and
fixed in sublimate-formaline (9 : 1). The adrenals were taken for histological

examination.

ADENOMATA IN THE EAT HYPOPHYSIS

The histology of the hypophyses

The hypophyses were imbedded in paraffin and sectioned, beginning from
the inferior surface and continuing until the pars intermedia and pars nervosa
were reached. The sections were distributed among four microscope slides, of
which one was stained by Crossmon’s (1937) modification of the Mallory stain,
one by Gomori’s (1950) aldehyde-fuchsin, and two by the McManus periodic
acid-ScIiiff (PAS) method as modified by Purves and Griesbach (1951a). In the
latter investigations we have used instead of Gomori’s (1950) formula, a stable
aldehyde-fuchsin powder^’ prepared according to Gabe’s (1953) method. This
powder (0-5 g.) was dissolved in 100 ml. 70 per cent alcohol, filtered and acidified
with 2 ml. of concentrated hydrochloric acid. This solution may be used
immediately after preparation and keeps for 4-6 weeks at room temperature.

Before staining, the sections were oxidised rvith Lugol’s iodine solution for
20 minutes. Permanganate oxidation, as used by Gabe (1953), destroys the
specificity of the staining of the basophil cells of the rat pars anterior.

In general the sections were cut at 5 p tliickness, but a certain number were
alwaj’^s taken at 2-5 p tliickness. These tliin sections were used to obtain photo-
micrographs shovdng satisfactory detail. For visual examination the 5 p.
sections were generally preferable because of the stronger staining.

All the sections were searched systematically on the mechanical stage of a
binocular microscope and the number and type of tumours found in each
hypophysis recorded.

Tumours were recognisable as areas composed of a single cell type with signs
of displacement and compression of the surrounding tissue. Eiddence of
cellular abnormality was present in all the nodules classified as tumours.

Uniformly large or uniformly small cell size, excessive variability of cell size,
excessive variability of nuclear size and chromatin content and the presence of
abnormal mitotic figmes were considered to indicate cellular abnormality. The
absence of hyalinisation in basophil tumours in gonadectomised animals in which
normal gonadotrophs were hyalinised was also considered an indication of
abnormality.

Tumours composed of cells containing glycoprotein granules as revealed by
PAS staining were classified as basophil tumours. These were subdivided on
the basis of the response to aldehyde-fuchsin staining. Tumours whose granules
were stained by aldehyde-fuchsin were classified as beta cell tumours and those
whose granules were not stained by aldehyde-fuchsin were classified as delta
cell tumours. Tumours composed of cells which contained acidophil granules
(stained red by the Crossmon stain) and those whose cells contained no recognisable
specific granulation were grouped together in the acidophil-chromophobe class.

In general, sundval of the gonadectomised rats was good and most of the
animals were in a good state of health when killed.

The survival of adrenalectomised rats was, however, not good despite the
supper of saline as dnnldng water. Of the 60 rats which were adrenalectomised
m addition to gonadectomy only 22 sundved for the length of time necessary

W. E. GRIESBACH AND H. E. PUE^^S

for comparison with tiie non-adrenalectomised controls. The fatalitj'^ rate was
greatest at the age of 4-8 weeks when many of the rats died either suddenly or
more slowdy with oedema of the legs and fluid in the abdominal and thoracic
cavities. The Iddneys frequently showed pathological changes. In nearly all
the rats of this group who had lived for 18-24 months after operation, adrenal
cortical tissue without the medulla rvas found. In only three animals was no
adrenal tissue seen -by gross inspection of the region of the anterior pole of the
Iddney. It seems to be certain that the animals survived because adrenal
cortical tissue had been provided either by small remnants not removed during
operation or by accessor 3 ’’ adrenal tissue growth. The adrenal tissue found post
mortem never exceeded the size of a normal adrenal nor did it contain tumour
tissue microscopicallj’’.

Tumour incidence

The results are summarised in Table I. The results of both sexes have been
combined since in none of the groups was there au}'^ significant difference in the
incidence of tumours in the sexes. There ^vas no significant difference between
Group D receiving 0-25 mg. stilboestrol and Group E receiving 0-05 mg. stilboestrol.
Therefore the results of these two groups have been combined.

The main finding was a high incidence of delta cell tumours in all gonadecto-
mised rats that survived more than 15 months. This incidence was not markedly
affected by the age at which gonadectomy was performed, or by the other treat-
ments (stilboestrol implantation, adrenalectomy) wliich were combined with
gonadectomy in Groups I), E and I. Tins t 3 q>e of tumour is quite uncommon

Table I. — Adenomata in the Hijpophysis of Gonadectoimsed Eats

Age of rats
(montlis)

Group of

rats

start

death

Gonadeotomised

. U

19^254

. 3

22-35

, 9

28-36

D & E .

, 14

14^191

. U

191 — 2a4

■

194-254

Total gonadeotomised
Not gonadeotomised

■ H

. 14

114

. 14

174

• l|-

151-194

—

18-24

. —

161-17J

Total non-gonadectomised

Number
of rats

Additional

treatment

Total

With

Ade-

nomas

. 29

. 21

Stilboestrol

. 24

0-25 mg.

0 • 05 mg.

. 38

Adrenalectomy

142

132

Stilboestrol

. 24

0-25 mg.
0-05 mg.

. 15

0-05 mg.

Adrenalectomy

. 15

. 68

—

. 21

iing Gj)

135

Adenomata Analysis according to
found cell tj'pa

Per

Acidophil

hypo-

Chromo-

Total

physis

Delta

Beta

phobe

3-4 .

4-2 .

4-9 .

1-9 .

2-2

1-8 !

—

S95 a^

.2-8 .

344

1-3 .

46 a^

.1-1 .

ADBNOJ.tATA IN THE HAT HYPOPHYSIS

in non-gonadectomised rats. In 135 animals in Groups I?, G, J and K only 8
possible delta cell tumours were seen and, as explained later, of these, 3 tumours
contained little glycoprotein and were not easily classified.

Beta cell tumours were seen in 3 gonadectonused animals and / non-gonadecto-
mised animals. Gonadectomy, therefore, did not appear to produce or inhibit
the formation of beta cell tumours.

Acidoplul-chromophobe tumours were found in 48 animals of all groups
examined at ages over 15 months. Gonadectomy did not have any marked
effect on the incidence of tlois type of tumour.

Multiple tumours were frequently found. In general, the number of tumours
found per hypophysis increased with age.

Varieties of delta cell tumour

Altogether 344 delta cell tumours were seen in gonadectomised rats. After
studying these tumours we found that they could be allocated to one or other of
three fairly weU-defined groups. There were 61 tumours in 49 animals in which
the cells, by the coarsely-flocculent nature of their granulation, elosely resembled
normal peripheral gonadotrophs (FSH cells). These tumours we will call
“ peripheral gonadotroph-like tumours ” (Fig. 1, 3, 7, 9, 11, 13).

More prevalent were tumours composed of ceUs closely resembhng central
gonadotrophs. Altogether 241 “ central gonadotroph-like tumours ” were found
in 117 animals. In tumours of this group a more or less uniform dispersion of the
basophil granules resulted in a diffuse pink coloration of the cytoplasm. In
addition there might also be some coarse PAS positive granules adjacent to the
Golgi body but the appearance of the peripheral cytoplasm was characteristic.
As in normal central gonadotrophs the diffuse PAS reaction was often stronger
in the interior of the Golgi region and the negative image of the Golgi was, in
consequence, especially conspicuous (Fig. 2, 4, 5, 8, 10, 14).

Forty-two delta cell tumours were composed predominantly of lightly
granulated cells. In these ceUs the PAS reaction was usually confined to the
interior of the Golgi body, the rest of the cytoplasm being quite pale. Occurring
among cells of this appearance were always some ceUs with diffuse pink coloration
throughout the cytoplasm (Fig. 6, 12, 15, 16). Three delta cell tumours found
in non-gonadectomised animals were of this Ughtly-granulated type.

Distribution in the anterior lobe

The different types of tumour were not distributed at random within the
anterior lobe but showed specific preferences for certain situations. The
peripheral gonadotroph-like tumours were confined to the inferior surface and
the peripherj^ of the pars anterior, especially at the anterior border where the
portal vessels enter from the stalk. This distribution corresponds to the distribu-
tion of the peripheral type of gonadotroph. In contrast the central gonadotroph-
hke tumours were most often found in the anterior lobe at a point where the
portal vessels have broken up into normaUy-sized capillaries. Central
gonadotroph-hke tumours were not, however, confined to this region and were
found, though m lower numbers, in other regions with the exception of the

Wl,r« ' > “ ' ‘r”” '“"'y investigation.

U hen It nas found it was situated in the central area (Fig. 17) or posterior to the

W. E. EEIESBACH AND H. D. PUEVES

triangle formed by the pars intermedia. The acidophil-chromophobe cell tumours
had a preference for the posterior edge of the anterior lobe. They were never
seen in the area where the large portal vessels enter the anterior lobe.

The stmcture of the adenomata

In most adenomata a well-defined tissue structure was developed. This
usuallj'’ took tlie form of a grouping of the cells into small rounded masses
enclosed by basement membranes as in the normal pars anterior tissue. In
some tumours this arrangement was more regular and well-defined than in the
normal rat pars anterior in wliicli the arrangement is somewhat irregular. In
other tumours a well-differentiated structure was present which differed from the
normal structure of the pars anterior. In these tumours cords and sheets of
cells were present, separated from each other by irregular blood-filled spaces
lined bj'^ endothelium (Fig. 5, 14, IS). An absence of any formed structure
(anaplasia) was found most common^ in the acidophil-chromophobe group of
tumours and was associated with the more extreme degrees of cellular abnormaUt}^
which were common in tliis gi-oup.

Abnormalitj’’ of the blood vessels was common in both basophil and acidophil-
chromophobe tumours. Greatly dilated rmssels and large blood-filled spaces of
irregular shape were often seen in both groups. Only in the acidopliil-chromo-
phobe group were haemorrhagic tumours seen, but in tliis group almost all
tumours showed haemorrhagic areas or signs of old haemorrhage in the form of
macrophages containing altered blood pigment. It is noteworthy that the
accumulations of pigment in the macrophages give an intense coloration with the
PAS reaction. These cells are easily distinguished from basophil cells by the
bromi colour of the pigment.

Evidence of secretion by acidophil-chromophobe iumonrs

In 20 of the 48 gonadectomised animals with acidophil-chromophobe tumours
there was evidence of lactogenic hormone secretion. The mammary glands
showed extensive development apparent to naked-eye examination, and evidence
of milk secretion was seen. In some there were milk-filled C3''sts and dilated
ducts from which milk spurted when the glands were cut. In others liistological
examination showed dilated ducts filled noth eosinophilic secretion. These
effects were found in 10 males and 10 females ; the mammary changes appeared
the same in both sexes. Secreting mammary tissue was not found in association
unth any of the acidopliil-cliromophobe tumours in the non-gonadectomised
groups in this series.

Adrenal changes in gonadectomised rats

In all gonadectomised rats an unusual degree of hyperaemia was found,
especially in the reticular zone of the adrenal cortex. The blood vessels in tins
region were widely dilated, forming large blood-filled spaces (Fig. 19, 20). A few
small adenomata were seen in the cortex but none resembling the large tumours

described by Houssay ei uL (1955). r .n n i

There was no evidence of secretion of oestrogen or androgen from the aortal
cortex or tumours arising therefrom in any of our gonadectomised amnials. e
seminal vesicles and prostates of all tho males were atrophic, and the uten 0 the

ABKNOiMATA IN THE RAT HYPOPHYSIS

females were small and nnstimnlatcd. Only in some females implanted with
stilboestrol pellets and killed one to two months later were enlarged fluid-filled
uteri found, an effect which is ascribed to the exogenous oestrogen.

Phaeochromocytomas were quite frequent in the adrenal medullae of the rats
examined but are not considered to be relevant to this investigation.

DISCUSSION

The significant finding in these experiments is a high incidence of basophil
tumours vuth the staining properties of delta cells in all groups of gonadectomised
rats which survived in good condition for 15 or more months. The production
of these tumours must be ascribed to the continuous maximal stimulation of
gonadotrophs by gonadal hormone deficiency. We have already referred to the
fact that two tjqies of gonadotroph can be distinguished — a peripherally-situated
type which we believe secretes FSH and a centrally-situated type which we
beheve secretes LH. It is noteworthy that the majority of delta cells tumours
in gonadectomised rats could be classed as resembling in cytological appearance one
or other of the two normally occurring gonadotrophs. Also the tumours of the
two types were differently distributed in the anterior lobe, the distribution of
each type being similar to the distribution of the normal gonadotrophic cell which
it most resembled and from which it was presumably derived.

In the bat {Myotis myotis) Herlant (1956) found that distinctive FSH and LH
secreting cells could be easily differentiated by staining reactions. Unfortimately,
in the rat the properties of the granules of the two types of gonadotroph are so
similar that we have been unable to distinguish between them by staining reations,
although a solubility difference in the granulation has been shown by Barrnett,
Ladman and MacAllaster (1955). We have not, therefore, been able to show that
the two varieties of delta cell tumour which we have seen contain granules of a
different quality.

The lightly-granulated delta cell tumours warrant some discussion. From the
fact that cells of this appearance can be found in tumours predominantly composed
of central gonadotroph-like cells and that occasional well-granulated cells can be
found in tumours predominantly formed of lightly-granulated cells we are
satisfied that these tumours are basophil tumours. It should be noted that the
tendencj'^ to retain glycoprotein granules within the Golgi body is a characteristic
of normal central gonadotrophs under degranulating conditions. It is our
opinion that most of the lightly-granulated delta cell tumours, in which the PAS
reaction was confined mainly to the interior of the Golgi body, were central
gonadotroph-like tumours in which differentiation was more marked than in

the easily recognisable central gonadotroph-like tumours. The tendency for
PAS positive material to be confined to the Golgi region was associated with a
greater degree of pleomorphism of the cells, and in many cases it appeared that
an area composed of cells of this form had developed in an adenoma of the typical
central gonadotroph-like type. Such appearances are interpreted by us as an
indication that lightly-granulated tumours are often derived from typical central
gonadotroph-hke tumours by an additional neoplastic change.

Woolley and Dickie (1949), as well as Houssay et al. (1955) have stressed the
fact that m their gonadectomised animals hypophysial adenomata were found
only m ammals which had neoplastic changes in the adrenal cortex and they

W. B. GRIESBACH AND H. D. PUR\rES

EXPLANATION OF PLATES

Fig. 1. Low power view of a basophil adenoma in the pars anterior of a rat killed 27 months
el k T ^ situated at the periphery of the pars anterior and the

PAS X ° normal penpheral gonadotrophs. Note also the dilated blood vessels.

Fig. 2.— Low power view of a basophil adenoma occupying a central position in the pars
anterior of a rat killed 191 months after gonadectomy. The cells of the adenoma contain
varj'ing amounts of granulation. The tissue structure of the adenoma is similar to that of
normal pars anterior tissue. The cells resemble normal central gonadotrophs. PAS x 60.

anterior of a rat killed 32 months after gonadectomy.
On the right is a well-granulated basophil adenoma whose cells resemble peripheral gonado-
trophs. Slightly to the left of centre is a basophil adenoma of moderate granule content,
composed of cells resembling central gonadotrophs. PAS X 60.

Fig. 4. — Low power view of a basophil adenoma in the pars anterior of a rat killed 27 months
after gonadectomy. Cells of moderate granule content, resembling central gonadotrophs,
form the left portion of tlie adenoma. The right side of the adenoma is composed of cells
of low granule content, the PAS reaction being confined to the Golgi region. PAS X 60.

Fig. 5. Low power view of an adenoma in the central region of the pars anterior of a rat

killed 19 months after gonadectomy. The tissue structure is abnormal, being composed of
strands of colls separated by blood-filled spaces which are white in the photo-micrograph.
The cells of this adenoma resemble central gonadotrophs. PAS X 60.

Fig. G. — Low power view of an adenoma in the central region of the pars anterior of a rat

killed 24 months after gonadectomy. The cells of this adenoma are basophils of low-

granule content, the PAS reaction being, in the main, confined to the Golgi region of the
cell. Tissue structure is abnormal, consisting of well-defined cell cords separated by blood-
filled clefts (white in the photomicrograph). PAS X GO.

Fig. 7. — High power view of peripheral region of the pars anterior of a rat killed 3 months
after castration showing the characteristic features of peripheral gonadotrophs affected by
castration changes. Note the coarse, strongly PAS positive basophil granulation, A
large proportion of the colls are hyalinised forming tj^iical “ signet-ring ” cells. The
negative imago of the Golgi body is visible in some of the cells as a white ring or crescent.
PAS X 400.

Fig. 8. — High power view of the central region of the pars anterior of a rat killed 3 months
after castration. As compared irith Fig. 7 the central gonadotrophs are seen to have a
more uniformlj^ distributed, finer granulation, giving the cytoplasm a pink colour in the
PAS stained section and appearing grey in the photomicrograph. The negative image of
the Golgi body is visible in most of the cells. In the hyalinised cells the granulation still
retains its diffuse appearance. PAS X 400.

Fig. 9. — ^High power view of the cells of a basophil adenoma in the peripherj' of the pars
anterior of a rat killed 24 months after gonadectomy. The cells have a resemblance to
unhyalinised peripheral gonadotrophs. PAS X 400.

Fig. 10. — High power view of the cells of a centrally-situated basophil adenoma in the pars
anterior of a rat killed 29 months after gonadectomy. The cells resemble unhyalinised
central gonadotrophs. Note the prominent negative images of the Golgi bodies and the
even distribution of fine granulation through the cj’toplasm. Tissue structure is normal.
.PAS X 400.

Fig. 11. — High power view of part of the adenoma shown in Fig. 1. The cells resemble
unhyalinised peripheral gonadotrophs, with a reduced granule content. PAS X

Fig. 12. — High power view of part of the adenoma shown in Fig. 4. The region shoum

includes cells resembling central gonadotrophs in the left of the field and, to the right, cells

of low-granule content. The dark spots in these cells are due to PAS staining of the cyto-
plasm within the Golgi region. PAS X 400. , j oa tv,

Fig. 13. — High power view of a basophil adenoma in the pars anterior of a rat k^ed 24 mimtns
after gonadectomy, shmving cells with a marked resemblance to unhyalinised penpheral
gonadotrophs. PAS X 750.

Fig. 14. — High power view of the basophil adenoma of Fig. 5 shelving the resemblance ot tne
cells to unhyalinised central gonadotrophs. PAS X 750. , 4 „a + 1 .=

Fig 15.— High power view of a basophfi adenoma in the pars anterior of a rat killed _4 momns
after gonadectomy. The cells are variable. A few are strongly granulated. Many are
lightly granulated rounded cells resembling central gonadotrophs. Others have pale pen-
pheral cytoplasm with a strong PAS reaction within the Golgi region. PAS ^ '

Fic 16.— High power view of a basophU adenoma in a rat killed 24 months after :

The ceUs are lightly granulated wdth the PAS reaction confined, m the rnain, to the koigi
regions which appear as black dots in the photomicrograph. PAS X 330,

British Journai. of Cancer.

Vol. XIV, No. ].

Griesbach and Purves.

British Journal of Cancer.

Vol. XIV, Xo. 1

ADENOMATA IN THE EAT HYPOPHYSIS

therefore assumed that oestrogens or androgens of adrenocortical origin -were
imphcated in the formation of the hj^ophysial adenomata. Dickie and Lane
(1956), however, found in one strain of inbred mice that hypophysial adenomata
appeared earlier and in liigher incidence than adrenal neoplasms. We have tried
to approach this problem by adrenalectomising our rats in combination \yith
gonadectomy. The attempt to keep adrenalectomised rats for the long-time
interval, necessary for hypophysial adenoma formation, has failed through high
mortality as well as through regrowth of cortical adrenal tissue. It was observed,
however, that regrown adrenal tissue never exceeded the size of the normal adrenal,
that it was usually found on one side only and that it never contained neoplastic
structures as judged by microscopical examination. We are certain that in the
surviving twenty-two adrenalectomised and gonadectomised rats no adrenal
tumour can have influenced the formation of hypophysial adenomata. Hjrpo-
physial adenomata were found in these animals in nearly the same number (1-8
adenomata per hypophysis) as in the rats only gonadectomised (2-2 adenomata
per hypophysis). The supply of minute amounts of oestrogens or androgens by
adrenal cortical tissue could not be excluded in our adrenalectomy series. The
fact should be stressed, however, that not a single animal of the 142, gonadecto-
mised for 18-32 months, showed signs of stimulation of either the uterus or the
seminal vesicles and prostate. The secondary sex organs without exception were
atrophic. Bielschowsky (private communication) working with the same strain
of rats has made the same negative observation. Nevertheless, we have
investigated the influence of small doses of stilboestrol in pellet form on the
production of pituitary adenomata. In gonadectomised animals the oestrogen
did not accelerate the formation of tumours or increase the number found. The
higher dose of oestrogen (0-25 mg. stilboestrol) may have stimulated the formation
of the acidophil-chromophobe type of tumour in intact rats since the incidence
of this type of tumour was significantly higher in Group F than in the untreated
animals of Group K (P < -01).

On the evidence existing at the present time we do not believe that in
gonadectomised rats sex hormones are indispensable for the initiation of hypo-
physial adenomata, although the possibility of oestrogen production in adreno-
cortical tumours in some strains of mice and rats is not denied (Woolley 1958).

It does not appear that the age of the animal at the time of gonadectomy
affects in any way the subsequent course of hypophysial changes resulting
eventually in delta cell tumour formation. In an earlier study (Purves and
Griesbach, 1955) we concluded from the examination of a small number of

Fig. 17.— Low power view of the pars anterior of a rat killed 17 months after gonadectomy.
In the upper half of the field is a delta cell adenoma, the granulation of which is unstained
In the lower half of the field is a beta cell adenoma, the granulation of which is stained bv
aldehyde-fuchsin. AF. — Orange G. x 72. ^

Fig 18.— Low power view of the pars anterior of a rat killed 19 months after gonadectomy
showing an acidophil adenoma composed of well-granulated acidophil cells Mall x 72 ’
Fig. 19.— Low power view of the adrenal cortex of a normal rat for comparison with Fig 20
G, zona glomcrulosa ; P, zona fasciculata ; K, zona reticularis; M, medulla. H. & e!

Fig 20.— Low power view of the adrenal cortex of a rat kUled 19 months after gonadectomy
showing changes seen in all long-term gonadectomised rats in this series. Note the widel^
H & E reticularis and the inner part of the zona faLculata

W. E. GBIESBACH AND H. D. PUEVES

gonadectomised rats that the stimulation of the gonadotrophs by gonadectomv
continued at a high level for only four to six months and that, thereafter the
activity of these cells diminished. It, therefore, seemed that some additional
factor, such as secretion from adrenal tumours, might he necessary for lone-
continued stimulation of gonadotrophs and eventual tumour formation after
gonadectomy. We now believe that no factor other than gonadal hormone
deficiency is necessarj^ for these effects. The regression of the castration changes,
which we previously observed after long-term gonadectomy, we now ascribe to
the effects of intercurrent infection, particularly to a chronic lung disease prevalent
in our rat colony at that time. Earl}'^ in the present study we noticed that rats
which had been unhealth 3 '’ for some time before death would show a regression
or an absence of castration changes in the hjqiophjmis, while in healthy members of
the same group the castration changes would be present at full intensitJ^

The acidophil-chromophobe group of tumours included a few with well-
gi’anulated acidophil cells which would be easily recogm’sed as an acidophil
tumour. In most of these tumours only small amounts of granulation were
present. The tumoiirs, therefore, were wealdy stained and appeared pale and
relativelj'^ chromophobic as seen at low magnification. Under high power
examination the presence of small numbers of acidophil granules in manj”^ of the
tumour cells could he seen and occasional enlarged cells of Ifigh granule content
were found. The presence of these granules characterises the cells of these
tumours as belonging to the acidophil class — i.e., as being cells the storage form
of whose secretion, when present, is the acidophil granule. We have called
these tumours acidopliil-cluromophobe to indicate that wlule the tumours may be
predominantly clmomophobic thej'’ are composed of lightly granulated and
degranulated cells of the acidophil class. The somewhat greater incidence of
acidophil-chromophobe tumours in gonadectomised animals {M per cent), as
compared with the non-gonadectomised groups (22-9 per cent) cannot be regarded as
significant. It appears that these tumours occur spontaneous^ in old animals,
and the greater incidence in the gonadectomised rats might be ascribed to a
greater average age in these animals. It is, however, noteworthy that gonadec-
tomy does not hinder the appearance of tumours of tliis tj^e, nor does it prevent
lactogenic hormones being secreted in amounts wliich have striking effects on
the mammary gland. It appears that mammary development and lactation
are indeed much more likely to occur in gonadectomised animals bearing such
tumours than in non-gonadectomised animals. Although not seen in tliis series,
mammary growth and lactation can occur in intact animals bearing spontaneous
acidophil-chromophobe tumours, one such case occurring in a female rat having
been observed in our colonjn

SITJUIAKV

A high incidence of basophil adenomata was found in the l^poply'^ses of rats
of both sexes examined 15 months or more after gonadectomy. No factor other
than a long-continued deficiency of gonadal hormones in an otherwise ea y
rat was necessary for the induction of adenomata. .

The cells of basophil adenomata induced by gonadectomy had the staining
reactions of delta cells and in most tumours resembled in cytologicaJ features
the normal gonadotrophic basophils from which they were eviden ly ern •

Functioning acidophil tumours secreting lactogenic hormone were requen

ADENO>LATA IN THE RAT HYPOPHYSIS

gonadectomised animals. Similar manifestations of lactogenic hormone secretion
were much less common in old, normal animals hearing acidophil tumours.

REFERENCES

Baeenett, R. J., Ladmax, A. J. axd McAluaster, X. J. — (1955) .7. Histochem.
Gytochem., 3, 391.

Bielschowsky, F. — (1955) Brit. J. Cancer, 9, 80.

Ceossmoe, G. — (1937) Anat. Bee., 69, 33.

Dickie, M. jM. akd Lake, P. W. — (1956) Cancer Res., 16, 49.

Gabe, M. — (1953) Bull. Micr., appl., 3, 152.

Gomoei, G. — (1950) Amer. J. din. Path., 20, 665.

Geiesbach, W. E. ak'D Pceves, H. D. — (1956) Proc. Univ. Otago med. Sch., 34, 1.
BLalah, N. S. — (1950) Endocrinology, 47, 289.

Heelakt, M. — (1956) Arch. Biol., Liege, 67, 89.

Houssay, B. a., Houssay, A. B., Caedeza, A. and Pento, R. M. — (1955) Schweiz med.
Wschr., 85, 291.

Peeves, H. D. — (1956) Proc. R. Soc. Med., 49, 1014.

Idem AND Geiesbach, W. E. — (1951a) Endocrinology, 49, 244. — (19516) Ibid., 49, 427.

—(1951c) Ibid., 49, 652.— (1954) Ibid., 55, 785.— (1955) Ibid., 56, 374.

Woolley, G. W. and Dickie, M. M. — (1949) Cancer Res., 9, 373.

Woolley, G. W. — (1958) Ciba Colloquia on Endocrinology, 12, 122. London (J. & A.
Churchill).

HISTOLOGICAL AND HISTOCHEMICAL CHANGES INDUCED BY
^KYLATING AGENTS IN TRANSPLANTED RAT SARCOMA
WITH SPECIAL REFERENCE TO SARCOLYSINE

M. A. PRESNOV

From the Department of Experimental Gliemotherapjj of the Institute of Experimental and
Chmcal Oncology. Academy of Medical Scie7ices of the U.S.S.R., Moscoiv.

Received for publication January 20, 1960

Since substances ivitli aiitineopJastic activity became available, particular^
those knoAvn as biological alk 3 'lating agents, sj’^stematic histological and cyto-
logical studies of their action have been made (Y'oshida, 1952 ; Jdanov, 1955 ;
Koller and '^^'eronesi, 1956 ; Kellner, 1957 ; Breuvis, 1958 ; KoUer, 1958 ;
Larionov and Presnov, 1958; Presnov and Spasskaya, 1959).

In this report the histological and histochemical changes that occur in several
experimental tumours following the administration of particular alkylating
agents are described.

MATERIALS AND METHODS

Three transplanted rat tumours were used.

Sarcoma 45. — This is a rat tumour of Russian origin. It is a malignant
spindle-ceh tumour, transplantation of which is successful in 100 per cent of
animals, and ndheh Icills its host in 25 to 30 daj'S. This tumour regresses
completely with sarcolj^sine, dopan and ethjdene-imino-benzoquinone (E39).

Sarcoma iIf-1. — This is a malignant, poljTOorphous-cell tumour of Russian
origin. On transplantation 100 per cent of grafts are successful, and the tumour
lulls its host in 30 daj^s. Sarcoma M-I is more resistant to chemotherapy than
Sarcoma 45.

Yoshida sarcoma . — Tliis well-known tumour was first introduced into the
Soviet Union in 1957.

All three tumours were transplanted subcutaneouslj’’ into albino rats of
either sex weighing between 80 and 100 g. The Yoshida sarcoma was also
studied in the ascites form. 633 rats were used.

Chemotherapy was begun when the transplanted tumours were 7 to 10 days
old, measured 0-7 to 1-0 cm. in diameter, and weighed 0-5 to 1-0 g. In the case
of the ascites tumour, treatment was begun 4 daj’^s after transplantation.

Tliree alk 3 dating agents were used : sarcoL'^sine, p-di-( 2 -chIoroeth 3 d)amino-
DL-phen3dalanine, (Larionov, Kliokhlov, Sclikodinskaya, Vasina, Truscheikina,
Novikova-Smirnova, 1955 ; Truscheikina, 1956 ; Larionov, 1959) knovm as
merphalan in Great Britain (Bergel and Stock, 1953, 1954 ; Bergel, Burnop and
Stock, 1955) ; dopan, 5-di-(2-cIiloroethyl)amino-4-methyl-uracil (Larionov and
Platonova, 1955 ; Platonova, 1957 ; Larionov, 1959) ; ethylene-imum-benzo-
quinone, E.39, 2, 5-diethylenimino-3,6-dipropoxy-l,4-benzoquinone (Domagk,

Peterson, and Gauss, 1954 ; Domagk, 1958).

CHANGES INDUCED IN RAT SARCOMA BA" SARCOLYSINE

Rats bearing Sarcoma 45 received either sarcolj^sine or dopan ; those bearing
Sarcoma M-1 received only sarcolysine, wliile rats bearing the Yoshida tumour
received each of the three compounds. The doses and routes of administration of
the drugs are shomi in Table I.

Table I. — Doses and Routes of Administration of Alkylating
Agents in Transplaled Rat Tumours.

Tumour Sarcolysine Dopan E3!)

Sarcoma M-1 . Intraperitoneal . — •

5 mg. per kg. 5 to 7
times, 3-daj' inter-
vals

10 mg. per kg. 5 to 7
times ot 3-* or 7 -day
intervals

Sarcoma 45 . 15 mg. per kg. once . Oral . —

or in 3-5 divided 0 • 3 mg. per kg. daity

doses 5 mg. per kg. for 15 da 3 'S, or O' 75

at 3-day intervals mg. per kg. 7-9 times

everj’ 3 daj’s

Yoshida solid . Intraperitoneal . Oral . Intraperitoneal

l'5mg. per kg. daily 0-3 mg. per kg. daily 0- 6 mg. per kg. daily

for 20 days for 20 days for 20 daj's.

Yoshida ascites . As above, or orally . — . —

* Some rats died.

The histological and histochemical methods used were as follows :

(a) ParafiSn-embedded material fixed in 10 per cent neutral formol saline.
Haematoxylin and eosin, Peulgen’s method for deox 3 Tibonucleic acid, methyl-
green-pyronin for ribonucleic acid, Van Gieson’s stain for collagen. Foot’s stain
for reticulin, and toluidine blue for metachromasia.

(b) Frozen sections of material fixed in 10 per cent neutral formol saline.
Sudan III for neutral fat.

(c) Paraffin-embedded material fixed in cold 80 per cent ethanol. Gomori’s
method for alkaline phosphatase. The incubation time was between 2 and 5
hours (Presnov, 1954, 1956).

(d) Paraffin-embedded material fixed by Schabadasch’s method (1949).
McManus’s modification of Hotclildss’s periodic-acid-Scliiff (PAS) method for
polysaccharide.

(e) Methanol-fixed smears of ascitic fluid. May-Griinwald-Giemsa.

(f) Vital fluorescence microscopy was carried out in collaboration with Dr.
P. V. Breuvis to study the nucleoproteins.

To allow for artefacts in staining, the treated and untreated animals were
killed at the same time, and the tissues from both were handled side by side
throughout each procedure, so that every preparation from a treated tumour
could be compared directly with its counterpart from an untreated tumour which
had been subjected to identical manipulation.

KHiSULiXS

Sarcoma 45 regressed in every animal under treatment with sareolysine and
dopan, but Sarcoma xM-l regressed completely only after administration of

M. A. PKESNOV

sarcclj’^sine at high dosage (five to seven injections at 3-day intervals of 10 mg
per kg. of body AveigJit). Subcutaneous nodules of the Yoshida sarcoma regressed
completely under tlierapy noth sarcolj^sine or dopan in only 25 per cent of the
rats, and in onlj^ 12 per cent when E39 was given. Sarcolysine always caused
complete regression in the ascitic form of Yoshida sarcoma, whether oral or
intraperitoneal routes were employed.

The histological and histooheniical clianges during the course of regression
were similar in all three tumours and with each of the three drugs.

General histological changes . — During the course of regression, striking changes
were observed both in the parencliyma and stroma of the tumoms. The earliest
changes, decrease in mitotic activitj^ and nuclear fragmentation, were observed
vdthin 1 hour of the intraperitoneal injection of sarcolysine in the case of the
Yoshida ascites sarcoma, and in 3 hours in the case of the solid sarcomata, or in
the ascites sarcoma after oral administration, hlitotic activity disappeared if
treatment Avas continued. Cells in prophase AA^ere the first to disappear. Cell
destruction accompanied the decrease in mitotic activity, their nuclei became
angular and the chromatin coarsened. Pj^knotic changes were less common.
Abnormal multipolar mitoses appeared. This sequence of changes is illustrated
in Fig. 1-6, 16-24. A A'erj’^ characteristic change was the enlargment of many of
the cells, which has been frequently described as a feature of the action of alkylat-
ing agents. With further decrease in mitotic actiAuty, bizarre multinucleated
giant cells appeared, presuraablj’’ as a result of amitotic (fiAusion (Fig. 2, 6, 17, 18).
During treatment the nucleoli of the tumour cells increased in size, showed
increased pyroninoplulia and alkaline phosphatase activity.

The stromal and parenchymal changes described did not occur simultaneously
throughout the tumour but AA'ere patchy and irregular. In the Yoshida ascites
cells the changes Avere comparable AAoth those seen in the solid tumours, and
included the appearance of pathological mitoses, the formation of multinucleated
cells and marked enlargement of the cells and their nuclei and nucleoli (Fig.
22-24).

Clianges in the nucleoproteins . — ^The first change obserAi-ed was droplet formation
AAdthin the nuclei (Fig. 5). The droplets stained deeply Aidth Feulgen’s stain
and also showed alkaline phosphatase actiAuty. The nuclei of the greatly enlarged
cells wmre Feulgen negative almost always, but rarely garm a positive reaction.

Studies on the nucleoproteins in tliis material, by means of Autal fluoresence
microscopy shoAA^ed that changes occurred 3 to 6 hours after the first treatment
AAdth sarcolysine, and consisted of increased fluoresence in the nuclei of affected
cells (Breuvis, 1958).

Fatty change . — Fat is seen only in small amounts in necrotic areas of the un-
treated tumours, but appeared in the form of small droplets in the cytoplasm of
a majority of the cells 24 to 48 hours after the beginning of treatment. Later all
the cells contained fat, and the small droplets fused into larger ones, becoming
liberated in the stroma as cells disintegrated. EA'^entually large quantities ot
fat accumulated, and this could still be recognised embedded in the scar Avmch
remained after the tumour had regressed completely and had become replaced

by fibrous tissue. • i + a

Alkaline phosphatase activity . — ^In untreated tumours, the enzyme is loca e
mainly in the nuclei and nucleoli of the cells. Following treatment, it appeared
also in the cytoplasm, while in fragmented nuclei the amount increased great y.

CHANGES INDUCED IN RAT SARCOjMA BY SARCOLYSINE

Later alkaline phospliatase activity appeared also in the stroma and between the
newly formed collagen fibres (Kg. 7, 8. 9).

Ribonucleic acid. — ^PjToninophilia, sometimes after an initial increase,
gradually decreased in the tumour cells, and later disappeared entirely.

Metacliromasia . — Cjdoplasmic metachromasia with toluidine blue also disap-
peared during treatment.

Glycogen . — Glycogen is not present in untreated tumours, exeept in lympho-
cytes in the stroma, or in necrotic areas. Glycogen did not appear in the tumour
cells as a result of treatment, though pink droplets, not disappearing after treat-
ment vnth amylase, appeared (McManus-Hotchldss reaction). They were
presumably mucopolysaccharide in nature.

Stromal reaction . — The first change was oedema, which separated the tumour
cells slightly from one another. Untreated tumours contain very little collagen,
but argyrophilic fibres are numerous. During treatment, collagen fibres appeared,
became increasingly numerous and eventually formed, a dense scar. During
the same period, the argyropliilic fibres lost their affinity for silver stains, became
thicker and were presumably transformed into collagen fibres (Fig. 10-15).

DISCUSSION

Alkylating agents kill cells. Death may be rapid, and is shoivn by fragmenta-
tion of the nuclei, or slow, when death is preceded by the appearance of fat in the
cytoplasm, increase in alkaline phosphatase activity, disappearance of ribon-
nucleic acid and of chromotropic substances from the cytoplasm, and by enlarge-
ment of the nuclei and nucleoli. Amitotic division may be a form of dystrophic
change in wliich the cells, though damaged, can survive. Since large doses of
alkylating agents can cause complete regression of tumours, it is likely that all
the cells are damaged to some extent, and the type and degree of damage may
depend on the functional activity of the cell at the time of exposure, some states
of activity rendering the cell more vulnerable than others. The drugs studied
appear to affect the process of cell division and also to interfere with growth, the
latter effect being relatively small. Sarcolysine appears to suppress the growth
of cells more than dopan or E39.

The alkylating agents appear to act on the tumour cells immediately as shmvn
by the rapidity with wliich idsible changes in the cells occur, for instance in the
ascites tumours. That nucleoproteins are damaged directly is suggested by the
early occurrence of nuclear fragmentation, increased fluorescence and decrease of
mitotic activity. It is possible that the exact mechanisms vary from one alkylat-
ing agent to another. Powerful agents like sarcolysine might have additional
actions.

It is important to emphasize that the stromal reaction follows the changes
in the tumour cells, and is perhaps directed towards removal of the damaged
tumour cells. It is assumed that increased alhaline phosphatase activity helps
m removing fragmented ceU components rich in phosphoesters and nucleic acids

SUJBIARY

1. The histological and lustochemical changes accompanying
regression were followed in tlnee transplanted rat tumours.

drug-induced

M. A. PRESNOV

3. T]ie drugs used were the alkjdating agents sarcolysine (merphalan), dopan

and ethjdene-imino-benzoquinone (E39). ^ ’

4. Eleven histological and histochemical methods Avere used.

5. Strildng parenchymal and stromal changes Avere observed. Early changes
included decrease in mitotic activity and nuclear fragmentation, Avhile later
enlargement of surviAung cells, and the appearance of multinucleated forms AA'as
seen. Fat appeared in the cjdoplasm, and ribonucleoprotein and chromotropic
substances disappeared. In the stroma, the argjwophilic fibres became conAmrted
into collagen. Alkaline phosphatase actirfity increases first in the cells, and later
in the stroma.

6. The alkjdating agents appear to act immediately on the tumour cells. The
stromal reaction is secondarj^ to the death of the tumour cells. The drugs afiect
the nucleoproteins dix-ectly.

I AAUsh to thanlc Professor L. F. Larionov for helpful adAuce.

REFERENCES

Bekgel, F. and Stock, J. A.— (1953) Hep. Brit. Bmp. Cancer Gampgn., 31, 6.— (1954)
J. chein. Soc., 2409.

EXPLAXATION OF PLATES

Fio. I. — Snrcomn 45. Untrented tumour. Hnemntoxylin and eosin. x280.

Fic. 2. — Sarcoma 45. 24 hours after the 3rd intrnperitonenl injection of sarcolysine, 5 mg.

per kg. of body weiglit at 72-liour intervals. Haematoxylin and eosin. x280.

Fig. 3. — Snrcomn 45. 72 hours after the 3rd injection. Hnemntoxj’Iin and eosin. x280.

Fio. 4. — Sarcoma 45. Untrented. Feulgcn reaction. x2S0.

Fig. 5. — Sarcoma 45. 48 liours after sarcolysine, 5 mg. per kg. of bod 3 ' weight. Feulgen

reaction. x2S0.

Fig. G.— Sarcoma 45. 24 hours after the 3rd intrnperitonenl injection of sarcolj'sine, 5 mg.

per kg. of bodj’ weiglit at 72-hour intervals. Feulgen reaction. X280.

Fig. 7. — Snrcomn 45. Untreated. Gomori reaction for alkaline phosphatase. x280.

Fig. 8. Sarcoma 45. 24 hours after the 2nd intraperitonenl injection of sarcolj'sine. Gomori

reaction. x280.

Fig. 9. — Snrcomn 45. 72 hours after the 3rd injection. Gomori reaction. x280.

Fig. 10. — Snrcomn 45. Untrented. A’^nn Gicson. x280.

Fig. 11. — Sarcoma 45. 72 hours after the 2nd intraperitonenl injection of sarcolj’sine. A'^an

Gieson. x280.

Fig. 12. — Sarcoma 45. 72 hours after the 3rd intraperitonenl injection. A'^an Gieson. X 280.

Fig. 13. — Sarcoma 45. Untreated. Foot. X2S0.

Fio. 14. — Sarcoma 45. 24 hours after the 3rd intraperitonenl injection of sarcolj’sine. Foot.
X ^80

Fig. 15. — Sarcoma 45. 72 hours after the 3rd intrnperitonenl injection. Foot. x280.

Fig. 1G. — Sarcoma M-1. Untreated. Haemntoxj’lin and eosin. x280.

Fig. 17. — Sarcoma M-1. 48 hours after 2 injections of sarcolj’sine, 10 mg. per kg. of body

weight at 73-hour intervals. Haematoxj’lin and eosin. x280. ri,j.

Fig. 18. — Sarcoma M-1. 48 hours after 5th injection of sarcolj’sine, 10 mg. per kg. ot body

weight at 72-hour intervals. Haematoxj’lin and eosin. X 280.

Fig. 19. — Sarcoma M-1. Untrented. Feulgen. x280.

Fig. 20. — Sarcoma M-1. 48 hours after the 3rd injection of sarcolj’sine. Feulgen. X-oU-

Fig. 21. — Sarcoma M-1. 48 hours after the 5th injection. Feulgen. x280.

Fig. 22. — V^oshida ascites tumour. Untreated. Maj'-Griinwald-Giemsa. x630.

Fig. 23. — A^oshida ascites tumour. 24 hours after the intraperitoneal injection of sarcolysine,

1 • 5 mg. per kg. of bodj' weight. Maj’-Grunwald-Giemsa. X 630.

Fig. 24. — V'eshida ascites tumour. 24 hours after 3 dnilv intraperitoneal injections ot sarco-
lj'sine, 1 • 5 mg. per kg. of bodj’ weight. Maj’-Griinwald-Giemsa. X 630.

British Joihinai- of Cancer.

Vol. XIV, Xo. 1.

Bresnov.

Bkitish Journal op Cancer.

Vol. XIV, Xo. 1.

Presnov.

CHANGES INDUCED IN RAT SARCOMA BY SARCOLYSINE

Idem, Buenop, V. C. E. and Stock, J. A. — (1955) Ibid., 1223.

BREuns, P. V.- — ^(1958) Arhli. Pat., 20, 39.

Domagk, — -(1958) Ann. N.Y. Acad. Sci., 68, 1197.

Idem, Peterson, S. and Gauss, W. A. — (1954) Z. Krebsforsch., 59, 617.

Kellner, B. — (1957) Acta Morph. Acad. Sci., Hung., 7, 4.

Koller, I?. C. — (1958) Ann. N.Y. Acad. Sci., 68, 783.

Idem AND Veronesi, U. — -(1956) Brit. .7. Cancer, 10, 703.

Jdanov, G. L. — (1955) Probl. Oncol., 6, 94.

Larionov, L. F. — (1959) Acta Un. int. Gancr., 15, 42.

Idem, Khokhlov, A. S., Schkodinskaya, E. N., Vasina, 0. S., Truscheikina, V. I.,
Koitkova-Sjurnova, M. A. — (1955) Bull. exp. Biol. Med., 1, 48.

Idem AND Platonova, G. N. (1955) Probl. Oncol., 5, 36.

Idem AND Presnov, M. A. — (1958) Arkh. Pat., 20, 32.

Platonova, G. N. — (1957) Bull. exp. Biol. Med., 6, 53.

Presnov, M. A. — (1954) J. gen. Biol., 5, 321. — (1956) Probl. Oncol., 4, 423.

Idem AND Spasskaya, I. G.— (1959) Ibid., 7, 38.

ScHABADASCH, A. L. — (1949) ‘ Proljlems Connected with the Histochemical Invebtiga-
tion of Glycogen in the Normal Nervous System. Studj'^ on the Biological
Characteristic of and Differences Between Neurones ’. Moscow (Medgiz).
Truscheikina, V. I. — (1956) Probl. Oncol., 2, 222.

Yoshida, T. — (1952) J. nat. Cancer Inst., 12, 947.

GEAFT-'SrERSUS-HOST REACTIONS IN THE RABBIT

K. A. PORTER

From the Department of Pathology, St. Mary's Hospital Medical School, London, 6^.2
Received for publication December 1, 1!)59

Until recent]}'^ tlie lioiiiograffc reaction was mainly studied from the aspect
of rejection of tlie foreign tissue transplant by its host. Yet, as Medawar (1958)
has pointed out, grafting is an act of parabiosis, however unequal the partners to
the union may be, and the possibilit 3 ' of the transplant mounting a counter-
attack against the recipient must be considered.

It was the ivork of Dempster (1953) and Simonsen (1953) on Iddnej^ homotrans-
plants in dogs that first indicated that reactions of tliis nature might be more than
a theoretical hazard. Since tiien the grave consequences of graft intolerance of
the host have been recogiused following induction of tolerance in newborn animals
by the injection of adult spleen cells (Billingham and Brent, 1957 ; Simonsen,
1957), following treatment of lethall}’^ X-irradiated animals ndth homologous hone
marrow (Trentin, 1956 ; Gongdon and Urso 1957). and following injection of FI
hj’^brid mice u’itli adult spleen cells from the parental strains (Cole and Ellis,
1958; Trentin, 1958; Gorer and Boj'se, 1959). In all these instances the injected
mice are for one reason or another unable to destroj^ the introduced foreign fym-
phoid cells which then proliferate and proceed to assail the host cells.

Although it is generallj’^ agreed that the victims lose weight and characteris-
ticallj'- show atrophy of their Ijmiphoid tissue, fev' detailed descriptions of the
pathological appearances associated with these syndromes are available.

The purpose of this report therefore is to describe and compare the histological
changes seen in rabbits suffering from secondarj'^ irradiation disease and runt
disease.

ilLVTERIALS AND METHODS

Animals .. — Young adult Chinchilla rabbits which were not inbred in the
genetic sense were used throughout this stud}'.

Production of radiation-chiinaeras. — Lethaily X-irradiated rabbits were treated
intravenous^ with bone marrow cells from normal adult rabbits,

1. X-irradiation . — Whole bodj^ X-irradiation was given as a horizontal beam
to 2 animals at a time from a Westinghouse machine under the following condi-
tions ; 220 kV., 12-5 mA., 70 cm. target to skin distance, 1-0 mm. Gu and 1-0 mm.
A1 filters, H.V.L. 1-8 mm. Cu, dose rate to sldn 33-4 r per minute, to centre of
animal 21 -1 r per minute. The dosage was divided ; an initial dose of 600 r at
the centre of the animal was followed 24 hours later b5'’ 500 r, and after another
24 hours by a further 500 r, giving a total of 1600 r (L.D. 100/30 days).

2. Bone marroio. — X suspension of adult homologous bone marrow ivas pre-
pared from a female rabbit as described earlier (Porter and Murray, 1958), and
1200 (X 10®) nucleated cells injected into the marginal ear vein of an irradiated

GEAFT-VERSUS-HOST REACTIONS IN THE RABBIT b"

male rabbit 1-3 hours follo^ving the last dose of X-rays, and -svithin 1 hour of the
death of the female donor.

3. Cvitcvion foT success . — ^The successful establishment of a radiation-chimaera
was accepted only if female type heteropliils (amphopluls) appeared in the irra-
diated male rabhit (Porter, 1957a.).

Induction of immunological tolerance

Spleen cells from adult homologous female rabbits were injected into foetal
rabbits (Porter, 1960a).

1. preparation of spleen cell suspensions . — A mature female rabbit weighing
2-5-3-0 kg. was anaesthetised ndth intravenous Nembutal and a splenectomy per-
formed through an upper midline abdominal incision. Immediately after removal
the spleen was finely chopped under aseptic conditions in heparanized saline
solution and then suspended by gently drawing up and down through a wide bore
cannula. After filtration through nylon bolting cloth of 90 p porosity a cell count
was made and a suitable quantity of the spleen suspension containing 50 ( X 10®)
nucleated cells injected into the recipient animal.

2. Injection of foetal rabbits . — A rabbit at the 20th or 22nd day of pregnancy
(normal gestation period is 31 days), was anaesthetised with intravenous Nembutal
and a midline abdominal incision made extending from the level of the last pair
of nipples to just below the umbiheus. When the abdomen had been explored
to determine the number of foetuses present, the free end of one horn of the uterus
was delivered through the wound. The position of each foetus was easUy seen and
its head and back identified by palpation. Adult spleen tissue suspension was then
injected through the wall of the uterus into the peritoneal cavity of each foetus.
After returning the uterus to the abdomen by gentle steady pressure, the peri-
toneum and hnea alba were sutured as one layer with 3-0 chromic gut. The
skin was sutured separately.

3. Skin grafts . — When possible the recipients of spleen cell suspension were
later challenged with skin grafts from the spleen donor. These were full thickness
homografts 2 cm. in diameter sutured into prepared beds upon the ears as des-
cribed by Stark, Brownlee and Grunwald (1958).

Weight . — The rabbits were weighed daily for the first month, then at weekly
intervals.

Antibiotic therapy . — All irradiated rabbits were given tetracychne hydrochlor-
ide intramuscularly 50 mg. per day for the first 2 weeks following irradiation.

Pathology . — All animals that were killed or died were examined post mortem.
A pencil of marrow was dissected from the right femur, the sample always being
taken from a comparable position at the mid-point of the shaft. Most tissues were
fixed in formol-saline, but the marrow, lymph nodes and spleen were fixed in
Helly’s fluid and all were routinely stained with haematoxylin and eosin. Special
stains were used where necessary, and marrow smears were stained by Leishman’s
method.

Sex chromatin was also sought in lymphocytes obtained from the lymph nodes
and spleen of some animals, using the method outhned by Riis (1957). The cell
suspension to be examined was incubated for 24 hours in an homologous plasma
coagulum and then appropriately fixed and stained. The disadvantages of this
technique have already been discussed (Porter, 19606),

K. A. POETER

Experimental procedure

In this experiment 4 groups of animals were studied.

Group I consisted of 5 normal adult non-irradiated rabbits and 14 baby rabbits
of various ages. Animals from this group were killed at appropriate times for
comparison uith those that had died or been killed amongst the treated groups.

Group II was composed of 20 X-irradited rabbits given no subsequent treat-
ment. They were examined histologically after death.

Group III consisted of 20 radiation-chimaeras with “ secondary disease ”,

In the accumulation of this group all successful radiation-chimaeras that were
regaining the weight lost immediately after irradiation, were subjected to biopsy
of an inguinal or axillary lymph node at 14 days after marrow treatment. After
this time anj’’ animal that continued to show a constant percentage of poIjTuorphs
wdth female sex chromatin, and that was clinically free of infection, but started
to lose weight again, Avas regarded as deAmloping “ secondary disease ”. Taking
the day AA^asting AA'as first noticed as the beginning of “ secondary disease ”, groups
of 3 animals Avere Idlled on the 1st, 7th, 14th and 21st day of the process for histo-
logical e.vamination. The remainder were examined AA'liencA’^er they happened to
die.

Group IV was composed of 20 non-irradiated tolerant rabbits AAuth “ runt
disease

In allocating animals to this group those rabbits subjected to intra-uterine
homologous spleen injection that failed to gain weight properly after birth, were
considered to have “ runt disease ”. The majority died aged 2-6 weeks and were
then examined histologically. In addition groups of 2 animals were killed and
examined at interAmls of 1, 2 and 3 weeks after birth.

RESULTS

Rabbits exposed to lethal X-irradiation tvithout marrow treatment [Group II)

All rabbits in this group had died bj”^ the 10th day, the mean surAUval time
being 8-1 ± 1A2 days. At post mortem some slight wasting was always present
and the fur often appeared dull and tended to be shed easity.

Bone marrow. — There was destruction of practically aU the haemopoietic cells
of the femoral bone marroAV, only the reticular cells surviAmig.

Spleen.— Tins organ was smaller than normal. The mean splenic weight being
0-22 ± 0-062 g. per kg. of body weight compared AA-ith 0-55 ± 0-046 g. for a group
of 20 normal rabbits. The decrease in size was due to almost complete destruction
of the lymphoid tissue. The reticular cells in the white pulp remained. The red
pulp was often congested and always contained haemosiderin, much AAotlun

macrophages. i

lApnphoid tissue elseioliere.—The thymus showed destruction of cortmai Jjnn-
phocytes -with shrinkage of the lobules and contraction of the stroma to form a
solid sheet of cells in the region where the cortex had been. Similar destruction
of lymphocytes with no evidence of regeneration was seen m the mesenteric,
axillaiy and inguinal lymph nodes, and in the normal bmrphoid
the gut, particularly the appendix. In 8 cases many red cells were present in the

lymph node sinuses.

GRAFT-VERSUS-HOST REACTIONS IN THE RABBIT

Besjnraiorti si/sicm. — A necrotic and liaeniorrhagic non-purulent infection of
the lungs by Ps. pifocnaneus, Icilled 6 of the animals in this group. Eight animals
showed intra-alveolar haemorrhage without infection.

Gastro-inlesihial tract. — Petechial haemorrhages into stomach and elsewhere
in the gut were seen in 16 animals. Rupture of the fundus of the stomach caused
the death of 4 animals and massive haemorrhage into the colon the death of 4
more. In 3 rabbits that died before 7 daj'S tliere was a partial denudation of the
intestinal mucosa ; the intervening areas consisting of flattened villi covered by
stretched epithelial cells. In the remaining rabbits, regeneration was complete
bj^ the time the}' died from infection or haemorrhage.

Liver. — In 6 cases there was some ati'ophy of the liver cells and distension of
the sinusoids w'ith blood ; T) more animals showed changes usually associated with
chronic venous congestion.

Testes. — Active spermatogenesis had ceased.

Ro other characteristic lesions w'ere noticed.

Radiation-chimaeras with “ secondary disease ” (Group III)

The animals with successful marrow transplants initially lost weight, but this
was followed by recovery from about the 10th day after irradiation. However,
in those developing “ secondary disease ”, at any time between the 16th and 40th
days weight loss recurred, accompanied by early signs of diarrhoea. These symp-
toms became steadily more severe until the animal died. At post mortem the
rabbits were invariably emaciated. Their fur was dull, dirty and shed easily.

Bone marrow. — All the animals, at w'hatever stage of “ secondary disease ”
they W’ere examined, show'ed a well-repopulated bone marrow (Fig. 1). Smears
revealed the presence of “ drumsticks ” in some of the cells indicating that re-
population was from donor sources. Animals killed or dying after the 14th day of
“ secondary disease ” showed loss of normal fat and increased csllularity. The
longer the disease had been present, the more pronounced this hyperplasia, which
was predominately of the myeloid series. Plasma cells were rarely seen in the bone
marrow. Erythropoiesis and megakaryocyte formation appeared to be normal.
A few phagocytic cells containing haemosiderin were always to be seen. In 12 of
the animals the stroma consisted of mucoid material which stained positively rvith
mucicarmine.

Spleen. — First day of “ secondary disease ”. In the 3 animals killed at the
beginning of “ secondary disease ”, the spleens appeared slightly larger than
normal. fiCcroscopically there was good repopulation of the white pulp with lymph-
ocytes, immature plasma cells and some transitional cells, using these terms as
defined by Fagraeus (1948). Mitotic figures were frequent, and small germinal
centres were present in 2 of the animals. The red pulp contained about as much
haemosiderin as was present in the control irradiated animals of Group II.

Seventh day of “ secondary disease ”. In animals killed at this stage the
spleens were enlarged, the mean weight being 1-00 i 0-183 g. per kg. of body
■"eight (normal = 0-55 :£ 0-046 g.), and microscopically the white pulp contained
many transitional cells, immature plasma cells and a few mature plasma cells and
^miphocytes (Fig. 2). Again mitotic figures were common. In 1 of the animals
lese cells were also found diffusely scattered throughout the red pulp. The
congested red pulp contained plenty of haemosiderin, mostly in macrophages,
rythrophagocytosis was present in all 3 spleens.

K. A. PORTER

Fourteenth day of “ secondary disease The 3 rabbits killed at this time still
showed shghtly enlarged spleens with a mean weight of 0-58 ± 0-03 g perks of
body Aveight Microscopically in some of the lymphoid nodules were cells ^th
pyknotio or fragmented nuclei, and others which had undergone complete necrosis.
Of the surviving cells a high proportion were mature plasma cells (Fig. 3). Other
l 5 nnphoid nodules in the white pulp had already been severely depleted of cells
and partlj”^ replaced by masses of smudgy fibrinoid material (Fig. 4). In one of
the animals foreign body giant cells were associated until this fibrinoid substance.
The red pulp was congested and now appeared to contain more haemosiderin than
was present in the spleens of the control irradiated animals.

Twenty-first day of secondary disease The spleens at this stage appeared
shrunken lyith a mean weight of 0-29 ± 0-03 g. per kg. of body weight. Micro-
scopicallj'' in 1 animal the white pulp had largelj'^ been replaced bj’' immature
collagen and numerous foreign body giant cells (Fig. 5). Lj^mphocytes, transi-
tional cells and immature plasma cells had disappeared, but the red pulp contained
scattered groups of mature plasma cells. Other animals examined at this time
showed similar loss of lymphoid tissue and substitution of fibrous scars for Mal-
pighian bodies, hut without giant cells (Fig. 6). Scattered groups of matiue
plasma cells were, however, always present and in 2 animals small haemorrhages
were also seen.

Tu'o rabbits that died 30 and 34 da 5 's after the onset of “ secondary disease ”
both showed extensive replacement of the depleted white pulp with mature
collagen. In all instances the red pulp contained much haemosiderin.

Other lymphoid tissue . — When biopsied at 14 days after bone marrow treatment,
the inguinal or axillarj’^ Ijraph nodes of those radiation-clumaeras wliich subse-
quentlj’’ developed “ secondar}’- disease ”, were small and showed early incomplete
repopulation with Ijmiphocjdes (Fig. 7). In cell suspensions from a few of these
lymph nodes female sex chromatin was demonstrated in l 3 fmphoc 3 des by the
method of Riis (1957).

the time the first animals udth “ secondary disease ” were killed the
mesenteric and other lymph nodes were enlarged and showed an extensive pro-
liferation of transitional cells and immature plasma cells, udth relatively few
mature plasma cells and Ij'mphocjdes. Ljmrphoid tissue elsewhere showed similar
changes, but the thjmius tended to lag in this process.

Little change was seen in the liistological picture obtained at the 7th day of
“ secondarj'^ disease ”, but bj'^ the 14th day many of the cells in the repopulated
lymph nodes rvere undergoing necrosis (Fig. 8) and mature plasma cells were
beginning to predominate amongst the surviving cells (Fig. 9). By the 21st day
the tymph nodes were slrrunken, depleted of lymphoc 5 des, and only contained a
sprinkling of mature plasma cells (Fig. 10). L^'^mphoid tissue elsewhere vas

similarly atropliied. • i, j r

Respiratory system.— In 6 of the 8 radiation-chimaeras that perished trom

“ secondary disease ” the immediate cause of death was a patchy purulent peri-
bronchiolar pneumonic consolidation. In 5 of these the organism responsible was

oistri-intestinal tract.— Of the 20 radiation-chimaeras with secondaiy
disease ” 4 animals showed discrete gastric ulceration. These
were about 0-5 cm. in diameter and situated at the pylorus. The high incidence
of this lesion, often accompanied by perforation, is weU recogmsed in the rabbir

GKAFT-VBKSUS-HOST KEACTIONS IN THE RABBIT'

after large doses of X-irradiation (Porter, 15)576). A colitis was present in 6
animals. This was most severe in the descending colon.

Liver . — Occasional focal areas of necrosis were present in the liver lobules of
4 of the S animals that died, and in tho.se of 2 of the animals killed at 14 days and
1 of the animals killed at 21 days. These lesions were scattered tlrroughout the
organ and were not consistently found in any one zone of the liver lobule. They
did not seem to be related to coccidial infection. Two of the 20 animals with
“secondary disease” showed biliary cirrhosis. This lesion has been described
previously (Porter, lOOOf/) and is usually associated noth I’ecrudescence of infection
with E. stiedae.

Testes. — Atroplij' of these organs was usuallj’^ present.

Amyloid was not found in these animals and no other characteristic lesions
were noticed. The skin appeared normal.

Rabbits suffering from “ runt disease ” (Grouj) IV).

All the animals in this group failed to gain weight at the normal rate. The
severity of the disease varied greatly, so that whereas a few of the baby rabbits
died within 15 daj's of birth following an “ acute ” attack, others suffered from
a “ chronic ” attack causing great retardation of growth and death 50-70 days
afterbirth. The mean survival time of those dying naturally was 31-93 ± 12-79
days.

Tliree runts were skin grafted from the spleen donor and proved to be fully
tolerant for as long as they" lived.

Bone marrow. — Of the 14 animals that died from “ runt disease ”, 3 showed an
aplastic bone marrow with loss of most of the haemopoietic cells (Fig. 11 and 12)
and 5 a femoral marrow greatly" depleted of cells (Fig. 13). These changes were
accompanied by" clinical evidence of immune haemolysis of the animals’ own red
cells and a steep fall in the leucocy'te and platelet counts (Porter, 1960c). The
other 6 animals showed in 2 cases a bone marrow of normal cellularity and in 4 a
granulocytic hyperplasia. Tlmee of the rabbits with normal or hyperplastic bone
marrow were males and smears showed occasional heterophils with “ drumsticks ”,
and similar female cells w"ere found in the peripheral blood, showing that some at
least of the bone marrow cells were derived from the spleen donor.

Of the 6 animals killed at various times after birth, the 2 examined at one
week both showed a bone marrow of normal cellularity in wliich female cells
could not be found ; the 2 rabbits killed at 2 weeks showed loss of cellularity of
he marrow ; and the marrow of 1 animal examined at 3 weeks -was aplastic,
Whilst the other showed masses of donor type heterophils and their precursors.

Rphen . — -In all 14 animals wth “ runt disease ” that died naturally, the
®P een appeared normal size in 8, and small in the remainder. hCcroscopicaHy"
lere was great diminution in the number of lymphoid cells in the splenic nodules
hn white pulp generally (Fig. 14 and 15). At best only a small halo of lympho-
} es and mature plasma cells was left around the splenic arterioles ; and in 6
finals the Malpighian bodies had been completely replaced by immature or
a ure collagen (Fig. 16). The red pulp was congested in many, and erythro-
P agocytosis was prominent in all spleens. Extramedullary haemopoiesis, although
P c-sent in some, was never very marked.

phoicT^ ^hbbits killed a week after birth showed apparently normal lym-

nodules in the spleen, but the other animal had an enlarged spleen with

K. A. POETER

many transitional cells and some immature plasma cells in the white pulp. Cell
suspensions from these spleens were examined by the method of Riis (1957), and
some mononuclears with female sex chromatin found.

EXPLANATION OF PLATES

Fig. L — Bone marrow from a rabbit killed 14 days after the onset of “secondary disease ”.
The marrow remains well repopulated, but there is loss of fat. Smears showed female cells.
H. andE. X 110.

Fig. 2. — Spleen from a rabbit killed 7 days after the onset of “ secondary disease ”. The
lymphoid nodule is well repopulated with transitional cells, immature plasma cells and a
few Ijunphocytcs. H. and E. x 110.

Fig. 3. — Sploeri from a rabbit killed 14 days after the onset of “ secondary disease Many of
the cells in the lymphoid nodule are necrotic. H. and E. X 50.

Fig. 4. — Sjileon from a rabbit killed 14 da3's after the onset of “secondary disease ”. The
Ij-mphoid nodule is depleted of cells and partlj' replaced bj' masses of smudgy fibrinoid
material. H. and E. X 50.

Fig. 5. Spleen from a rabbit killed 21 days after the onset of “ secondary disease . The

white pulp has been replaced b^' immature collagen and numerous foreign body giant cells,
H & 13 X SO

Fig. G* Spleen from a rabbit killed 21 days after the onset of secondarjr disease The

lymphoid tissue has disappeared and its place taken by fibrous tissue and a few scattered

plasma colls. U. and E. X oO. ^ r • i- j

Fig 7. Inguinal h'inph node removed at biopsy from a rabbit 14 daj^s after irradiation and

marrow treatment. Tliere is incomplete repopulation with lymphocytes. Female sex clwo-
matin was domonstratod in some of the ceUs from this node. ^ - ,,

Fio 8 —Inguinal Ivmplv node removed at post mortem from a rabbit kiJJed 14 days alter the
onset of ‘‘ seoondarj' disease ”. Note that many of the Ij^phoid ceUs have
necrosis. A biopsy of a node from the other grom of this animal, before the onset of
“ secondary disease ”, is shown in Fig. 7. H. and E. X 50.

Fig. 9.— Higher magnification of part of a mesenteric Ij-mph node from a rabbit killed 14 dajs
after the onset of “ secondary disease Mature plasma cells predominate amongst the

FirS -fiLtnierTc ly^fh nod^from a rabbit killed 21 days after the onset of “ secondary
disease ”. Ljunphocytes have disappeared, only reticular cells and a sprinkling of ma ur

Fig!° 1 L— FemS"Cne^al^ow fr^ a normal baby rabbit 30 days old for comparison ivith

Kig‘T2!-B?ne^maSowVom a baby rabbit that died from “ runt ®°;Sws

^'Ihore is almost complete | E ThO

“"ru^t di!eas^^when 40 days old There is
"^'^eat^pletion of im cells when compared with the marrow shown m Fig. 11.

Fi?’lL-§ieen from a nonnal baby rabbit 21 days old for comparison with Fig. 15. H. and

in a rabbit of this age. H. and E. X 50- ai^ease ” when 70 days old. There is loss

Fig 16 .— Spleen from a rabbit that died from runt disease wr^n v ^

J.,? friSlIiSi” ta » norS” tav’i.bbil 20 d.y. oM to oomp.ri.on .rill.

X 50. ^ rabbit 21 dai'S old for comparison with Fig. 20.

Fig. 19.— Appendix from a normal baby rabbit aujs

H. and E. x 50. . . jicease ” killed at 21 days. There is almost

this age. H. and E. X 50.

BltlTISH .ToUltNAI. or Canci'.u.

Vol. MV. No. I.

'.V' ?v;j..«‘ jt

Porter.

British Jourhai. of Ca>'cer.

Vol. XIV, No. 1.

Porter,

British Jouhnah of Cahcer.

Vol. XIV, No. 1.

11 12

Porter.

British Journal or Canchr.

Vol. XIV, Xo. 1

Porter.

GRAPT-^^SRSUS-HOST REACTIONS IN THE RABBIT

The spleens of the animals lulled at 2 weeks were normal size, but both micro-
scopically showed proliferation of transitional and immature plasma cells in the
wliite pulp with few lymphocytes and 1 rabbit showed in addition small patches
of fibrinoid necrosis in the lymphoid nodules.

The 2 animals killed at 3 weeks both had small spleens with great reduction in
lymphoid content of the white pulp and in 1 of the rabbits the splenic nodules
had been replaced by collagen.

Lymphoid tissue elsewhere.— M\ the animals that died from “ runt disease ”
showed, compared ndth controls of the same ages, shrunken lymph nodes con-
taining Very few lymphocytes (Fig. 17 and 18). This loss of lymphocytes was also
a striking feature of the intestmal lymphoid tissue, e.g., Peyer’s patches, and was
most ob\dous in the appendix (Fig. 19 and 20). The thymus was however, an
exception, for although there was undoubtedly some decrease in lymphocytes,
this was nowhere so obvious as in lymphoid tissue elsewhere.

In the animals killed at intervals after birth, primitive oells, including tran-
sitional and immature plasma cells, were numerous in the lymph nodes at 1 and
2 weeks, but by 3 weeks the nodes were atrophic. Lymphocytes were at all times
scarce.

Respiratory system. — ^The lungs of 12 of the 14 animals that died from “ runt
disease ” showed bronchopneumonic lesions at post mortem. In the 8 animals
with an aplastic or hypoplastic bone marrow tins was a necrotic and haemorrhagic
non-purulent infection, whilst in the remainder it took the form of a purulent
peri-broncliiolar consolidation.

Gastro-intestinal tract. — Apart from an ulcerative enteritis in 3 animals, no
other specific lesions were seen.

Liver. — In the livers of 6 of the 14 animals that died, and in the liver of 1 of
the animals killed at 3 weeks, there were small focal areas of necrosis. These
lesions were identical vath those seen in the irradiated rabbits suffering from
“ secondarj^ disease ”. Evidence of coccidiosis was sought but not found.

No renal or vascular lesions were observed in any of the animals. The skin
and other organs appeared normal. Amyloid was not found in any of the rabbits.

DISCUSSION

As the present study shows the pathological changes in “ runt disease ” are
very like those seen in “ secondarj-^ disease ”. In both cases the animals become
progressively more sick, waste and often develop diarrhoea. Death from infection
usually occurs some 2-6 weeks after the onset of the process. In both instances
by using sex chromatin as a biological marker it has been shown that the foreign
cells injected, or their descendants, persist and are present in the lymph nodes
and spleen, i.e., these animals are cellular cliimaeras. At first free proliferation
rapidly repopulates the host’s lymphoid system with poorly differentiated
pyronm-positive c^nor cells resembling the transitional and immature plasma
cells described by Fa^aeus (1948). After tliis initial increase there is a generalised
legression with atrophy of the new lymphoid tissue and its gradual replacement
bj collagen and scattered collections of mature plasma cells. Focal liver necroses
svn'droineT ' previously (Porter, 19606), are also a feature of both

K. A. POUTER

hen the haemopoietic tissues are considered, however, certain differenop,
are apparent between “ runt disease ” and “ secondary disease ” In “ seconS
Jsease ’’ there is rapid repopulation of the aplastic bone marrow, and even wh?n
the rabbit is dying this restored marrow always remains cellular and generallv
shows an extensive granulocytic hyperplasia. Use of the “ drumstick ” marker
shows that this colonisation is from proliferation of donor cells, and tins is con-
firmed by finding female type heteroplu'Is in the peripheral blood.

In runt disease , on the contrary, the bone marrow is frequently destroyed
and the animal develops a severe anaemia. Only in a few runts is this destruction
of host bone marrow accompanied b 3 '' progressive replacement -with haemopoietic
cells of donor origin. ^

Tliis difference is not difficult to understand when one remembers that in pro-
ducing radiation-chimaeras a ver^'’ large dose of haemopoietic cells and their pre-
cursors is given intravenously to an animal whose own bone marrow has been
destroyed by X-rays ; whereas in producing runts spleen suspension containing
relative^ few such haemopoietic cells is given intraperitoneally to an animal
whose own bone marrow is intact.

In both instances an immune haemolysis of host red cells is seen, accompanied
bj’’ a raised indirect serum bilirubin, eiythrophagocjdosis and a positive Coomb’s
test. Also in both, as the disease progresses, the peripheral blood lymphocyte
count fails steadily (Porter, 1960c).

These obsenmtions underline the essential similarity between “ secondai^"
disease ” and “ runt disease ” in the rabbit. Li both it seems the host is immuno-
logicallj^ defenceless : in “ secondarj’^ disease ” because of irradiation damage, in
“ runt disease ” because the immune system is insufficiently mature. The injected
foreign cells proliferate, invade, and repopulate the Ij’^mphoid system of the host.
Indirect evidence then suggests that thej^ proceed to attack the host cells, but how,
and whether tliis inv'ohms antibody, is not knoum. Purther, why histological
evidence of such an attack should be confined to haemopoietic, lymphoid and
possiblj'’ hepatic tissue, is equallj'’ obscure. As the postulated assault continues,
destruction of the restored Ijunplioid tissue also gradually occurs. At present, no
explanation of this secondarj'' loss of donor cells is entirely satisfactory. Kaplan
and Rosston (1959) suggest that the foreign cells probably die in the course of
killing host target cells : a process which they envisage may necessitate direct
contact between each donor cell and a very few target cells. It may even be that
the excessive amount of host antigenic material coming to the foreign lymphoid
tissue simply overvdielms and exhausts it. However, it is important to be clear
that no experimental precedent for such “ exhaustion ” exists. Whatever the
exact mechanism, it does seem that death of the chimaera is the outcome of
destruction of the antibody-producing cells of both host and donor.

As might be anticipated, giving irradiated rabbits homologous lymph node
suspension as well as bone marrow leads to rapid repopulation of the host s spleen
and Ijnnph nodes with foreign cells and the onset of an “ accelerated form o

“ secondary disease ” (Porter, 19606). „ , j w

Conversely, the incidence of “ secondary disease can be greatly reduced by
using foetal haemopoietic cells, which are Imown to be immunologically immatiire,
instead of adult bone marrow to produce the radiation-clumaeras (Uphott, ,

other examples of graft-against-host reactions have recently been recognised

GRAI’T-VEESUS-HOST REACTIONS IN THE RABBIT ‘o

and although, the details of each syndrome vary according to the circumstances of
the experiment, the end result is always wasting and lymphoid atrophy.

Thus, if splenic cells from parental strain donors are injected into hybrid
mice a wasting disease results which closely resembles secondary disease
(Trentin, 1958). In this instance the foreign cells are not rejected because the Fl
hybrid mice possess in their tissues all the antigens of both parent strains. The
injected lymphoid cells therefore proliferate and attack the host producing
histological changes very like those described in the present paper (Nowell and
Cole, 1959).

Similarly, many rat pairs placed in parabiosis with a cross-circulation develop
“ parabiosis intoxication ”, in which one animal remains well while the other
rapidly wastes and his lymphoid tissue atrophies. lATien such homologous rats
are separated after 5 days in parabiotic union a previously exchanged skin homo-
graft may persist in an animal which is dying with general lymphoid atrophy and
severe anaemia (Nakic and Silobrcic, 1958).

It thus seems that any transplantation of immunologicaUy competent cells
into an animal whose immime defences are for some reason paralysed, is poten-
tially hazardous and may, under conditions favourable to the invading cells,
produce a lethal wasting syndrome.

STUEMARY

Secondary irradiation disease and “ runt disease ” in rabbits are described
and compared.

Outstanding features of both are progressive wasting and diarrhoea, early
splenomegaly and enlargement of lymph nodes, followed later by shrinkage of
these organs and focal liver necroses. Overwhelming infection is usually the imme-
diate cause of death.

With the help of sex chromatin and “ drumstick ” markers, it is shown that
in both instances the animals are cellular chimaeras. There is early invasion and
repopulation of host lymphoid tissues by prohferating donor-tj^e transitional
cells and immature plasma cells. Later, these cells undergo necrosis and this is
sometimes associated vdth fibrinoid changes and foreign body giant cells. The
end result is extreme lymphoid atrophy with fibrosis.

The only histological difierence between the two conditions is that in
“ secondarj’- disease ” the aplastic bone marrow is rapidly colonised with donor
cells and is still liighly cellular when the animal dies, whereas in “ runt disease ”
the host’s bone marrow is frequently destroyed and less often is there repopulation
vlth donor-t}q)e haemopoietic cells.

The similarity between these syndromes and those known as “parabiosis
intoxication ” and Fl Itybrid “ wasting disease ” is noted and the conclusion
reached that they are all graft-against-host reactions.

^le author wishes to thank Mss Jane EendaU for valuable technical assistance
r. 1 1 Hulbert and Sister AA^oodward of the Radiotherapy Department, St. Mary’s
Hospital, for provic^g the X-irradiation facihties. and Lederle Laboratories
DiMsion, American ^anamid Company, Pearl River, New York, for generous
supphes of tetracycline hydrochloride. °

This work was supported by a grant from the Jledical Research Council.

K. A. POKTER

BEFERENCES

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Cole, L. J. and Ellis, M. E.— (1958) Science, 128, 32.

CONGDON, C. C. AND Urso, I. S. — (1957) Amer. J. Path., 33, 749.

Dempster, W. J. — (1953) Brit. J. Surg., 40, 447.

Fagraeus, a. (1948) Acta med. scand., Suppl. 204.

Gorer, P. a. and Boyse, E. A. — (1959) Immunology, 2, 182.

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Medaivar, P. B. — (1958) Proc. Roy. Soc. B., 148, 145.

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Porter, K. A. — (1957fl) Ibid., 4, 129. — (19575) Brit. .J. exp. Path., 38, 401. — (1959) Ibid.,
40, 273.— (1900«) Nature, Bond. 185, 789.— (19606) Clin. Radiol, 11, 22.— (1960c)
Ann. N.Y. Acad. Sci. (in press). — (1960d) Brit. J. exp. Path. 41, 72.

Ide/fi AND Murray, J. E. — (1958) J. vat. Cancer Inst., 20, 189.

Rns, P. — (1957) Nature, Bond., 179, 785.

SiMONSEN, 51. — (1953) Acta path, microbiol. scand., 32, 36. — (1957) Ibid., 40, 480.
St^irk, R. B., Brom’nlee, H. and Grunwald, R. P. — (1958) Aim. N.Y. Acad. Sci., 73,
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Trenten, j. j. — (1956) Proc. Soc. exp. Biol. N.Y., 92, 688. — (1958) A?w. N.Y. Acad.
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KIDlsTlY CAECINOBIAS OF THE EOYH. INDUCED BY THE
1 VTH9, eeticuloendothbliojia virus

J. G. CARR

From the British Empire Cancer Campaign Unit, Poultry Research Centre, Edinburgh
Received for publication December 18, 1959

It has been shown that the ES4 vims causing erythroleukaeniia of fowls
■would also cause renal carcinomas in veiy young chicks (Carr, 1956). It therefore
seemed opportune to re-examine the kidney tumours caused by the ]\IH2 ■virus
that ■u^ere described by Roulds (1934a, 19346). Though he concluded that there
is no reason to implicate the ^terable agents in their production he regarded
some as atypical metastases "with an epithelial element, saying of one that it
“ scarcely permitted any other name than adenoma (or adenocarcinoma) ’ , a
remark ■wliioh the illustration certainly appeared to confirm completely. Day-
old animals ■were sometimes used in his experiments, but no correlation of the
“ adenomas ” ■with the use of young hosts was mentioned, and his attempts at
transplantation of the adenomatous structures failed. He stated that the opinions
on their nature and production could only be regarded as provisional.

3IATEEIALS

The virus used in this work was revived from a ■vial of freeze-dried tumour
that had been prepared by Dr. P. R. Peacock at Glasgow over 18 years previously.
The fowls were all from the Centre’s flock of Bro^wn Leghorns.

METHODS

Virus preparations consisted of either a 10 per cent extract of macerated
tumour tissue in water, clarified by centrifugation at about 3000 g for 10 minutes,
or such an extract further purified by separation of the virus by high-speed
centrifugation and enzyme digestion (Bather, 1953). Cell grafts were made from
a 20 per cent suspension of tumour cells in saline which had been allowed to settle
for about 10 minutes. All injections were of 0-2 ml. injected through a very fine
(intradermal) needle.

KESUDTS

Alost of the kidney tumours were obtained in a similar way to that described
for the erjdhroleukaemia virus j decimal dilutions of a 'virus preparation were
injected intramuscularly into the legs of one or more groups of chicks of known
age, and the animals killed and examined as soon as the tumour began to interfere
vith their well-being. To these were added a few carcinomas encountered in
nrds injected uith an arbitrary dose of material in connexion ■with other experi-
ments, hut this was found to be a very inefficient way of inducing the kidney

shorr(5ehelo^) between inoculation and death was usuaUy too

J. G. CAJRE

Such investigations emphasised a very marked difference between the
reactions of birds of different ages to the primary inoculation, which will be first
described.

Growth of the primary tumour

There was a very obvious difference in the growth of the MH2 tumour,
whether originating from ceils or virus, depending upon whether the inoculation
was made into chicks less than one week old or into older birds. The usual slow
progression of the tumour as seen in birds of six weeks old or more, wdiich seldom
fills the muscle in less than 4-5 wmeks and often regresses, ehanged to a rapid
proliferation, which frequently inconvenienced the animal at about 14 daj^s.
Even when inoculated at the age of about nine days, tumour growth was slower
and they frequently regressed, so that titration by the methods of limiting dilution
was impractical at this age, while survival of younger chicks for the 26 da 3 '^s or so
that were found to be needed to produce kidnej’’ tumours in the case of the ES4
erjdhroleulraemia virus was likely to be rare. This difference was accentuated
by the variation in the metastatic spread of the tumours described in the next
section.

Dissemination of the tumour

The spread of the tumour cells m the body of the host was also sharpl}’’ different
in the young clucks. In birds 0 '\’’er 10 days of age metastases w'ere of moderate
frequency, usually seen as a few discrete white or j'^ellow^ sharply-circumscribed
nodules in the liver, and less frequently in the spleen, proventriculus and kidney.

In contrast the very young chicks often suffered a massive dissemination of
tumour cells which produced a striking resemblance to leukaemia. The liver and
spleen were often pale and grossly enlarged, due to a diffuse infiltration of malig-
nant cells whose mass sometimes was obviously greater than the primary. The
bone-marrow was similarly invohmd, and primitive cells were present in the
circulating blood. The general picture would certainly have led to the diagnosis
of leukaemia from the post-mortem appearance alone.

Intermediate stages, -with massive diffuse metastases into the viscera were
also often encountered.

Table I gives a summary of such an experiment using a rather heavy dose of
virus, which, despite the difference in age of the chicks of only 7 days, shows the
considerable difference in the reaction to the virus.

Table I. — Showing difference in Reaction to Virus at Two Ages

Age Primary SurvivaJ

(daj^s) Number tumour Leukaemoid (daj's)

1 . 8 . 8 . 6 . 11-21

8 , 8 . 4 . 0 . 19-28

The experiment was terminated and survivors killed at 28 days.

This association of a leukaemia-like condition with MH2 was also notiwd by
Foulds (1934) who reported that a “minority of birch inoculated with kIH2 die
of a disease which has some features in common with leukosis ”. The presen
finding, that tliis is confined to the very young hosts, was not mentioned by him.
The relation of this to leukosis is referred to again in the discussion ; it is mentioneU

KIDNEY CAECINOMAS INDUCED IIY VIRUS

here since, as will be realised, it contributed to the difficulties in obtaining the
kidney tumours.

Kidney groivilis

(а) Gross appearance . — In experiments wth older chicks, kidney involvement
was rather rare, in contrast to the youngest animals, which showed obvious
growths more often than not. These varied from a single lump which might
involve the whole of one kidney to over a dozen of assorted sizes in each organ.
Some, usually the larger ones, were solid and deep-ljnng, while others were
superficial and occasionally cystic. As Foulds noted, most of these were found
in birds survdving three weeks or more.

(б) Microscopic. — Histologically, these kidney tumours were of two distinct
kinds. One class was obviously a purely metastatic growth of ordinary MH2
cells, which started as an intertubular mass separating the tubules before their
invasion and destruction occured, as described by Murray and Begg (1930),
Foulds (1934) and others. Only this type was found in birds injected at an older
age, and in the younger animals which died early.

Contrasting with this was a kidney tumour confined to birds injected at the
age of 10 days or less — ^usually the groups inoculated at a few days — and surviving
for 26 days or more ; these limits being very similar to those found for the
erytbroleukaemia virus. These were, as Foulds remarked, frankly carcinomatous
growths of columnar eipthelium, and there seems no reason to avoid calling them
anything other than kidney carcinomas.

The small and early forms are of the type shown in Fig. 1. Ingrowths from
a simple cyst yield a papillomatous adenocarcinoma, whose central space is later
filled by the cancerous material to produce a picture of the type shown in Fig. 2.
Here the epithelial cells are forming multiple alveoli, which are sometimes filled
by necrotic material. These adenomatous or alveolar structures are separated
in places by a connective tissue stroma. Such growths may become very large,
replacing almost the whole of the affected kidney by a bulky tumour. Invasion
of adjacent viscera does not occur. The tumours have all had this rather simple
structure, and they do not show any of the complexities characteristic of embryonal
nephromas.

The epithelial cells usually contain a rather large and deeply-staining nucleolus
(Fig. 3), characteristic also of the sarcomatous form of the MH2 tumours. These
epithelial cells are extremely basophilic, much more so than the ordinary MH2
tumours. Fig. 1 shows an area of ordinary MH2 sarcoma metastasis adjacent to
a carcinoma whose very deeply stained cytoplasm makes the differentiation easy.

Small carcinomas have usually been found at the periphery of the organ
and like the erytbroleukaemia ones seem to have their origin in the nephrogenic
areas still to be found in this region. “

The results of a typical experiment are given in Table II. This was performed
on 3-day old birds, and the yield of three carcinomas out of 18 animals is rather
better than the average.

Transplantation

The conditions under which carcinomas were induced having been establit^Tiprl
.ttempt. were made to transplant them. Kidney growths oecJS™ ta Sue
birds A^ere selected, though of course it could not be certain beforehSd that they

J. G. CARK

Virus dose
(equivalent
tumour

Survival

Metastases
other than

Kidney

weight)

(days)

kidney

involvement

0-2 X 10-“

-f

+ +

-f-h

0-2 X 10-'

-1- +

-f- ~f-

-f- +

-h

0-2 X 10-“

-h-h !

+ +

—

0-2 X 10-“

+ +

—

0-2 X 10-’

* Means no primary

tumour induced.

Carcinonins

TJio experiment ires tenminnted find all animals killed at 33 dnj's.

were not merely metastatic sarcomas. One half of the tumour was used to prepare
histological sections, and the other half provided material for grafting into groups
of 4-12 birds. In practice, about half of those selected proved to be carcinoma-
tous. The results of these experiments can be summarised as follows :

Carcinomas were almost always transmitted to nearly all the hosts aged 1-18
days, but the same material would invariabl}' fail to produce anything but a
sarcoma in birds aged 30 days or more. In very young chicks the transplant
proliferated rapidty, and could incapacitate the cluck in about 15 days. The cut
surface of successfully-transplanted carcinomas u'as characteristic ; instead of
the plain waxy sirnface of the sarcomatous MH2, much of it showed a complex
pattern of whorls. Section showed areas of ordinary’' MH2 sarcoma mingled with
extensive carcinomatous areas of the t3'pe showm in Eig. 4. The proportions
varied, but it wms possible to get over 10 g. of transplant, the majoritj' of wliich
resembled this picture. Other tumours yielded onl}’’ a small central area ol
carcinoma, amounting to less than a gram in all.

As Fig. 4 indicates, the carcinomatous parts of these grafts did not usuall)
look as healthy as did the primary renal tumour, and gave the impression o
reverting to the sarcomatous structure. It was therefore not surprising tha
attempts to transplant these tumours for a second generation gave no convincing

EXPLANATION OF PLATE

Fia. 1 . — Showing early cystic carcinomas. Surface of kidney bottom right. Sarcomatoas
metastases bottom left.

Fig. 2. — Portion of large carcinoma.

Fio. 3. — Carcinoma, higher magnification ; note large nucleoli.

Fig. 4. — Transplanted carcinoma.

KIDNEY CARCINOMAS INDUCED BY VIRUS

evidence for the further proliferation of the carcinomatous portion, thougli it is
not yet clear whether this is due to reversion of the cells, death, or simple over-
grovdh by the sarcomatous portion.

Metastases m birds carrying carcinomatous transplants were always sarco-
matous in tjfpe.

DISCUSSION

These tumours are clearly analogous to the papillarj’^ adenocarcinomas induced
by the ES4: erjdhroleukaeniia virus (Carr, 1956) which are undoubtedly of renal
origin, and there can be no valid reason for not regarding and naming them as
such. Determination of their virus content was not made. Because of the
certam contamination by infective blood, and possible contamination by sarco-
matous areas as in Fig. 1, these could not be reliable and might be misleading if
any importance was attributed to the results. Foulds (1934) hesitated to consider
them as true carcinomas and referred to the work of Lauterberg (1919) on kidney
metastases in humans to explain the alveolar patterns in such tumours. The
frankly carcinomatous gi-owths do not, in fact, particularly resemble Lauterberg’s
description of such metastases, where the kidney tubules were replaced by
carcinoma cells, the glomeruli remaining intact, to give an architecture still
recognisable as kidney but whose tubules were made from cells of the primary
growth. The MH2 kidney carcinomas are formed by cells that are cytologically
distinct from the MH2 sarcoma cell of the primary tumour, as Fig. 1 and Fig. 3
show, and cannot be mere secondaries derived from it.

Definite proof of this contention is provided by the transplantation
experiments, where a suspension of cells sufficiently disaggregated to pass a fine
intradermal needle proliferated in muscle to give large areas recreating the original
pattern.

These transplantation experiments, if they thus aid in solving one problem,
pose a new one, that of the age limitation for successful transmission as a
carcinoma. Such a limitation for carcinoma, namely growth as a sarcoma in
older animals, but as a carcinoma in young ones, is unknown in other aspects of
cancer research. However, ivith this information gained with the MH2 virus, it
has been found that the ES4 erythroleukaemia virus carcinomas, previously
regarded as non-transmissible (Carr, 1956), can in fact be similarly transplanted
by cell grafts if very young hosts are employed (Carr, unpublished). A similar
factor may have been operating when Duran-Reynals (1946) only obtained sarco-
mas from fowl embryonal nephromas. If this change can be technically regarded
as an irreversible carcinoma to sarcoma transformation of the graft it is not at
first sight analogous ivith what is usually understood by this in the case of
mammalian tumours, though this may be in part a consequence of its great speed.
Comparable information of the age of the host in relation to this change does not
seem to be available for mammals.

The study of this aspect of the fowl carcinomas is rendered very difficult
because m each case virus liberated from the graft of the carcinoma will form a
sarcoma at the inoculation site, and it appears that this is the faster-growing
element. In the case of the older fowls, it is therefore not clear whether the
sarcoma wa,s derived from the carcinoma cells, or was induced by virus. Certainly
the grafts in young chicks appeared to be reverting by cellular change (Fig 4)
but even here this might be a reflection of the competition by the sarcomatous
8

J. G. CARE

elements induced on inoculation as these carcinomas are disintegrating in a host
younger than the original carrier.

The correlation of age of the Iiost and reaction to MH2 virus is indeed complex
as the work of Dhaliwal (1959) showed in the embryonic system. His anrlc
clearly proved that infection of the embryo cells by circulating virus was possible
without the necessity for injury. As in tlus work, it is very likely that the
leukaemia-like reaction of the young host is a true one, caused by infection of the
haemopoietic cells by circulating virus. It also seems likely that, corresponding
to the carcinomas, this is limited to a brief span of days, and then reverts to a
sarcomatous process. This equally makes definite proof of a true leulraemia
dilBcult, and is in part dependent upon the definition of leukaemia that is accepted.
At present, the condition is perhaps better referred to by a non-committal term
like “leukaemoid ”.

If some of these aspects appear Hi-defined and uncertain, this is a property
they share xvith the fowl leucosis complex to which they belong. The relation-
sliip of the “leukaemoid” reaction of MH2 birds to leucosis was earty recognised
by Houlds (1934) and is quoted above. The fowl leucosis complex has been held
to include Ij^mphoid, mymloid and erytlnoleukaemias, sarcomas, osteopetrosis,
osteosarcomas, fowl paratysis (neurolymphomatosis) and ocular lymphomatosis,
to which kidney carcinomas have been added by these investigations on the ESI
and MH2 renal tumours. For a recent review of this complex see Campbell
(1956). Few of these diseases liaAm had any detailed investigation of the poten-
tialities of their causative virus(es). On the other hand the relatively uncompli-
cated Rous 1 virus, which causes neither kidney carcinomas nor leukaemias in
young chicks (Carr, 1959) was shoum by Duran-Reynals (1947) to produce many
new varieties of cancer when modified by heterotransplantation. The complexities
of the work are, most probably, greater than has been appreciated, rather than
less.

StJMMAEY

The MH2 virus of reticuloendothelioma will also induce kidneys carcinomas in
very young chicks. These lesions are only found when the host survives for
several weeks, and seem analogous to those induced by the ES4 virus. They can
be transplanted for one passage to young hosts, but in older ones only sarcomas
result. The occurrence of a leukaemia-like condition with MH2 virus has been
confirmed. It is pointed out that it is also mainly a reaction of the young host
whose response to the virus changes rapidly'^ shortlj’^ after hatching.

All expenses in connexion with this work were borne by the British Empire
Cancer Campaign.

REFERENCES

Bather, R. — (1953) Brit. J. Cancer, 7, 492.

Cajupbell, j. G. — (1956) Vet. Bee., 68, 527.

Care, J. G.— (1956) Brit. J. Cancer, 10, 379.— (1959) Virology, 8, 269.

DHAunvAL, S. S. — (1959) Brit. J. Cancer, 13, 685.

Duran-Reynals, F. — (1946) Cancer Bes., 6, 645.— (1947) Ibid., 7, 99.

Foulds, L. — (1934(Z) Sci. Bep. Cancer Bes. FA, Bond., 11, 1. — (19346) Ibid., 11, o
Lauterberg, A. — [1919) Z. Krebsforsch.,\6, ^2. ^ 701

Murray, J. A. and Begg, A. M.— (1930) Sci. Bep. Cancer Bes. Fd, Bond., 9, i-

STUDIES ON MOUSE LEUKAEMIA. LEUKAEMOGENESIS BY
CELL-EKEE FILTEATES INOCULATED IN NEAVBOEN AND
ADULT JIICE

J. F. A. P. JnLLER

From the Chester Beatty Research Institute, Institute of Cancer Research :
Royal Cancer Hospital, Fulham Road, London, S.W

Received for publication December 11, 1959

Filtered extracts, usually from neoplastic tissues, can induce leulcaemias in
newborn or adult mice of certain strains (Gross, 1957a ; Graffi, 1957 ; Friend,
1957 ; Schwartz and Schoolman, 1959). Gross has shown that such extracts
from tissues of high-leulraemic strain mice with spontaneous lymphocytic leukae-
mias, w'hen injected at birth into mice of low-leukaemic strains, will ultimately
produce leukaemia in about a tliird of the recipient mice (Gross, 19586). The
resulting leukaemias were transplantable by cell graft in most cases to adult mice
of the recipient line but only rarely to the donor strain in which the origmal
leukaemia occurred.

Only newborn mice less than 16 hours after birth were found initially to be
susceptible to the leukaemogenic activity of the extracts. When, however, the
extracts were passed serially through several generations of susceptible mice, their
potency was increased and young adult mice could be successfully inoculated
(Gross, 1958c). Marked inter- and intra-strain sensitivity has been found in the
response of the mice to inoculation of the extracts. In the C3H strain, for instance,
the C3Hf/Gs subline is the most susceptible (Woolley and Small, 1957) while the
C3Hf/An subline is hardly sensitive (Gross, 1955a). The necessity of using new-
born mice and the marked differences in strain and substrain susceptibility have
suggested the possibility that the disease only develops in mice that can acquire
immune tolerance to the leukaemic agent (Harris, 1958 ; Barnes et al., 1959).

A series of investigations has been commenced in this laboratory in an attempt
to elucidate the mechanism of leukaemogenesis by cell-free extracts of leukaemic
tissues. The present paper reports the results obtained in four different strains of
mice inoculated as newborn or as adults with filtered extracts. A further paper is
concerned with the role of the thymus in leukaemogenesis by cell-free filtrates of
leukaemic tissues (Miller, 1960). A preliminary account of these studies has
already been given (Miller, 1959).

jMaterials and methods

Experiments were carried out on mice of four different inbred strains
(Table I). Inbreeding m mice of all four strains has been maintained by strict
sib-matings m our laboratory at Pollards Wood since the time of their acauisitinr
free ffltrafes .-ere prepared fern freshly obtaM by a

method closely follotvmg that of Gross (19586). The tissues were homogenized iu

J. F. A. P. MILLER

Table 1.— Source and Date of Acquisition of Inbred Strains

Strain

Date

acquired

C3Hf/PW

1938

C3Hf/Gs .

1958

CBA/H .

1949

Ak( .

1945

Source

Bittner.

Gross via Woolley.

Ham'ell.

Furth via Engelbreth-Holm.

ice-cold saline with a previously chilled mortar and pestle so as to make a 20 per
cent homogenate. This was centrifuged in a ServaU centrifuge at 1400 g for 15
minutes in the cold room. The supernatant fluid was then centrifuged at 7000 g
for 10 minutes and the final supernatant fluid filtered tlirough a chilled Selas 02
porosity filter candle under vacuum pressure. The extracts were either used
immediately or sealed in 2 ml. glass ampoules and stored at —79° C.

The extracts were prepared from thymus, spleen and lymph nodes of mice with
either spontaneous or induced leukaemias : the spontaneous leukaemias were
obtained from our own colony of Ak,- and from AI^R mice received from other
laboratories ; the induced leukaemias were obtained from C3Hf/Gs mice inoculated
by Dr. Gross with his potent Passage A filtrate (Gross, 1957h).

Inoculation procedures. — Filtrates were generally injected into newborn mice
less than 16 hours after birth, each baby mouse receiving O-l ml. by the intraperi-
toneal route, the needle first traversing the thigh muscles to avoid leakage,
Passage A filtrate was used however, suckling mice up to 14 days of age were
injected, the volume of the inoculum being varied according to age : at 1 and 2
days 0-1 ml. was given, at 3 and 4 days 0-2 ml. and at 5 to 14 days 0-3 ml. Adult
mice inoculated with ffltrates received 0-5 ml. intraperitoneally daily for 3 days.
Control mice were usually littermates identified by tad-clipping ; they received
either saline of filtrates heated to 56° C. for 30 minutes.

Transplantation of leuhaemias. — ^Por transplantation of leukaemias, cell sus-
pensions were prepared from fresh leukaemic spleen by teasing out with forceps
in a dish of saline. The cells were washed in saline to the desired volume so that
0-5 ml. of the suspension contained 30 to 50 million cells. This amount was
injected intraperitoneally into adult mice of the strains tested for transplant-
ability. The cells of each case of leukaemia to be transplanted were injected into
3 to 5 mice of each strain.

Induction of immune tolerance. — For induction of specific immune tolerance o
Ak tissues, cell suspensions from thymus or spleen of one-month-old healthy Ak,-
donors were prepared by teasing out in buffered Ringer phosphate solution,
washing twice, and resuspending in the buffered solution so that 0'05 ml. o e
suspension contained 5 to 7 million cells. This amount of the freslily prepare
suspension was injected into the anterior facial vein of newborn C3Hf/r n

less than 20 hours after birth. , j r u-irntr-

Shin grafts. — Skin grafts were performed according to the method or null g-
ham and Medawar (1951) at 6 to 8 weeks of age. The grafted skin was ^
months-old healthy Akf female mice and served as the external indicator ol me
tolerant state. Only fully tolerant mice, carrying healthy skin grafts were useu

experimentally.

Histology. — Tissues for histological examination were fixed in
stained in haematoxylin and eosin.

Bouin’s fluid and

LEUKAEMOGENESIS BY CELL-FREE FILTRATES

RESULTS

Detailed results are presented in Tables II to VIII. The salient features of
these tables may be summarized as follows.

Incidence of spontaneous leuTcaeinia (Table II)

Only the Alif strain had a high incidence of spontaneous leukaemia during the
fourteen month period of observation. The final figures for the full life span of
the low-leulraemic strains may well be rather different.

Table II. — Incidence of Spontaneous Lymphocytic Leukaemia
in Untreated Mice of Four Different Strains

Alice with

lymphocytic leuknemia

Strain

mice

Number of
mice observed
for 14 months

Number

Ago

in months

Per cent

C3Hf/PW

227

(12, 14)

Less than

C3Hf/Gs .

—

cba/h .

121

—

Ak, .

193

171

Average 8-9

Incidence of lymphocytic leukaemia in mice inoculated at birth with cell-free extracts
of leukaemic tissues (Table III)

(a) Controls. — The saline and heat-inactivated filtrate controls in aU cases did
not have a significantly different incidence of leukaemia from the completely
untreated mice.

Table III. — Incidence of Lymphocytic Leukaemia in Mice Following
Inoculation at Birth of Cell-Free Extracts

Filtrate
given at

Strain birth

f Ak leukaemic

C3Hf/PW

K Passage A

C3Hf/Gs

CBA/H

Ak(

[^Saline

{ Passage A
Heated Passage A
f Ak leukaemic
(^Saline
Ak leukaemic
J Passage A
Saline

Number

group

39
87t
15
71

40
75
15t
34

♦ S^'ivors are over 12 to 14 months of age.

T these mice wore injected 1 to 14 days after birth.

Mice with

lymphocytic leukaemia*

Age

Number in months

Per cent

7-14

11-8

(average 11)

7-10

26-7

(average 9)

—

2-4

100

(average 3)

—

6-15

(average 11)

—

3-6 (average 4*7) 1
7-12 ^

78-7

3-6

100

(average 4-1)

7-11

(average 9 • 6)

J. F. A. P. MILLER

(b) Filtrates from Ah leuhaemic tissues. — Filtrates from leukaemic Ak mice had
a marked effect only in Ak,- mice where the average age of onset of the disease was
reduced from about 9 months to about 5 months. Tins effect must be regarded
as an acceleration of the leukaemogenic process. The total incidence of the
disease in the inoculated group was slight^ lower than in the completely untreated
group as a result of a higher mortality from non-leukaemic causes in inoculated
mice.

A slightly increased incidence of leulcaemia was observed in C3Hf/PW and
CBA/H mice following injection of Ak leulraemic filtrates at birth.

Thirty-one separate Ak filtrates were used in these experiments but only S
were associated with leukaemogenic activity in 03Hf/PW and CBA/H mice
(Table IV). Five filtrates each produced leukaemia in 2 mice and in 3 cases the
2 mice came from the same litter. Sixteen of the filtrates used were responsible
for the development of early leulcaemias in inoculated Ak,- mice.

(o) Passage A filtrate. — Injection of Passage A filtrate at or soon after birth gave
100 per cent incidence of leukaemia in both C3Hf/Gs and Ale,- mice as early^ as 2 to
4 months. This filtrate increased the incidence of leukaemia following injection
into newborn C3Hf/PW mice to about 27 per cent the disease developing between
7 and 10 months of age.

Table — Activity of Filtrates from Tissues of Ah Mice with Spontaneous Leukaemia

Recipients (inoculated at birth)

Low-Ieukaemic C3Hf/PIV
and CBA /H mice

A ^

Number Leukaemia

High-leukaemic Akj mice

Number Number

incidence

Number

of mice

suscept-

^ ^

Per

of mice

Source of

active

inocu-

ible

active

inocu-

filtrates

lated

litters

Number

cent

filtrates

lated

Air, (Pollards AVood)

5/15

0/20

9-0

10/14

AKR (Manchester) .

1/8

2/10

6-3

3/5

AKK(Gif-sur-yvette) 1 /4

1/4

10-5

2/3

AKR (Paris) .

1/4

13-3

1/2

Total .

S/31

139

10/44

9-4

16/24

Number

suscept-

ible

litters

15/lS

3/5

2/3

22/29

Incidence of ecrl;
(3-G months)
leukaemia

Number

Per

cent

G7-5

C4-7

77-8

CC-/

68-0

Transplantability of the leuhaemias

Seven of the leulcaemias produced in C3Hf/PW after injection of ffltrates of
Ak leiikaemic tissues Avere tested for transplantability in Akj and CSHf/Pn nuce.
All were transplantable to C3Hf/PW and 3 to Ak mice as well.

Passage A induced leukaemias in C3Hf/Gs mice were transplanted, -ty
transplantable to CSHf /Gs and 1 5 also to Ale,-. Three leukaemias arising m OEA/n
mice AA^ere transplantable only to CBA/H mice.

The development of a potent Ah filtrate (Table V)

Filtrates from Ak leukaemic tissues was passed through serial cell-free in-
oculations into 4 successAe generations of newborn Ak mice. In eac i ’

the first Ak mouse that developed leukaemia was used as donor for the prepara

LEUICAEMOGENESIS BV CELL-FBEE FILTRATES

Table Y.—The Incidence of Lipnphoct/fic Leukaemia in CZHflGs Mice Following
Inoculation of Ah Leukaemic Filtrates Passaged Serially Through Ah Mice

Filtrate given
nt birth

Ak leukaemic (from siiontoneous case)
Ak leukaemic (after 3 passages in Ak
mice) _

Ak leukaemic (after 4 passages m Ak
mice)

Number of
C3Hf/Gs mice
inoculated

10 *

C3Hf/Gs mice with
Ijunphocytic leukaemia

r '

Ago

Number in months Per cent
0 — 0
0 4 — 10 40*5

(avorago 7-4)

11 3'G 08-8

(average S-l)

* These mice wore inoculated between 1 and 5 days after birth.

of the next passage-extract. The final filtrate considerably increased the total
incidence of the disease and accelerated the average age of onset when inoculated
into C3Hf/Gs mice at or soon after birth.

Table Yl— Incidence of Lymphocytic Leukaemia in Mice Following Inoculation

of Cell-Free Extracts

at i to 8

Weeks of Age

Jlice with

lymphocytic leukaemia*

Filtrate

” \

given at

Number

Age

strain

4 to 8 weeks

in group

Number

in months Per cent

C3Hf/PW

. Ak leukaemic

—

r Ak leukaemic

—

C3Hf/Gs .

J Passage A

4-8

(average 6-8)

Aki .

. Ak leukaemic

8-11

83-3

(average 8- 9)

Survivors are

over 12 to 14 months of age.

The incidence of lymphocytic leukaemias in mice inoculated as adults with cell-free
extracts of leukaemic tissues (Table VI)

Cell-free filtrates of leukaemic tissues of Ak mice were not capable of inducing
leukaemias in adult C3Hf/Gs or C3Hf/PW mice. Though a high number of Ak^
mice developed leukaemia following injection at 4 to 8 weeks of age with filtrates
of Ak leukaemic tissues, the total incidence and average age of onset were almost
the same as in untreated Ak^ mice. It must, therefore, be assumed that the
filtrates had no noticeable efltect when injected into adult mice.

Passage A filtrate given to 03Hf/Gs at 4 to 8 weeks of age did produce some
leukaemias but not as efficiently as the same filtrate given at or soon after birth.

The incidence of lymphocytic leukaemia in GSHfjPW mice tolerant to Ak^ and
inoculated as adults with cell-free extracts of Ah leukaemic tissues (Table VIII)

An intravenous injection of healthy Akj- spleen or thymus cells into newborn
C3Hf/PW mice induced long-lasting tolerance to Ak^ skin grafts in up to 90 per
cent of the mice (Table VII). Transplants of Ak leukaemic ceUs grew progr^s-
ively in all cases in tolerant C3Hf/PW mice but never in non-tolerant mice On
further transplantation to groups of non-tolerant 0311 and of Ak^ mice the trans-

J. F. A. P. MILLER

Table Yll.—Tolerance to Ah Skin Graft in GSHf/PW Mice
Inoculated at Birth with Ah Spleen or Thymus Cells

Ak cells given
nt birth

Mumbor Tj^pe

5-7 million Spleen

5-7 million Thjnnus

0 —

Number of
mice in
group
143
161
57

Number of mice
full}^ tolerant
128 (89%)*
137 (85%)*

0 (0%)t

* Ak skin graft intact to date (over 12 months),
t Air skin graft rejected in 1 1 ± 1 days.

plants were successfully established only in Ak mice thus showing that the genetic
constitution of the transplanted leukaemia was still Ale.

Table VIII. — Incidence of Lyniphocrjtic Leukaemia in CSHfjPW Mice Tolerant
to Ah Following Inoculation of Ah Leukaemic Extracts at 4 fo 6 Weeks of Age

Intravenous injection
at birth

Filtrate given at

4 to 6 weeks

Number
of mice
in group

Mice with leukflemm

f ^ ^

Number Age Per cent

Ak spleen cells

Ak leukaemic

0 — 0

Ait thj-mus cells

f* >«

0 — 0

Ak spleen or tlninu.s cells .

None

0 — 0

No leukaemia occurred in C3Hf/PW mice made tolerant to Ak and injected 1
month after birth Muth cell-free filtrates from Ale leukaemic tissues (Table VIII).
Sixteen of the mice in this group received at least one of the 8 filtrates that was
associated with leukaeniogenic activity following inoculation into newborn
C3Hf/PW or CBA/H mice.

DISCUSSION

The work described here confirms the results obtained by Gross (19586) and
others (Woolley and Small, 1956 ; Furtli et al., 1956 ; Dulaney et al., 1957 ; Hays
and Beck, 1958 ; Kassel and Rottino, 1959).

Cell -free extracts of tissues of Alt mice with spontaneous leukaemia are clearly
capable of leukaemogenic activity. In the high-leultaemia Ak strain and in the
(AKRXC3Hf )Fi hybrids, there is little or no change in leukaemia incidence hat
an acceleration of the onset of the disease following inoculation of extracts at
birth (Rudali, Duplan and Latarjet, 1957 ; Law% Dunn and Boyle, 1955). The
leukaemic agent has been shown to be present in young healthy^ Ak mice (Gross,
1951, 1953), so that inoculation of leukaemic extracts into these mice must be
assumed to increase the quantity of agent already present in the host. Accee-
ration of leukaemia may thus be explained on a purely quantitative btyis. Ex-
tracts from normal tissues of low-leultaemic C3H mice failed to be associated w i
leukaemogenic activity followdng inoculation into newborn CSH mice (Gross,
19556). These mice may, therefore, not carry the leukaemic agent, and leukaemia
occurring as a result of inoculation of Ak leukaemic extracts must presuma j
have been induced by the agent. It is true, however, that the disease occurs
spontaneously in low-leukaemic strain mice (Law, 1957) and two cases have een
diagnosed in our colony of C3Hf/PW kept under observation for only 14 mon is.

LEUlCAETilOGENESIS BY CELL-EREE EILTRATES

These two cases still have to be explained. It is possible that some of the CSH
mice carry the agent, but that our methods are not sensitive enough to demon-
strate its presence ? If this is so, does the inoculation of leukaemic filtrate at
birth simply accelerate the onset of the disease in mice that would develop it if
they lived long enough ? Some agent must be present in C3H mice since Gross
claims to have activated it by X-irradiation (Gross, 1958fl). The point at issue
is whether this agent is or is not identical to that jiresent in high-leukaemia strains.

Filtrates from Ale leukaemic tissues have been shoAvn to vary considerably in
their activity. Many of them were associated with the development of early
leukaemia in Ale mice, but only few produced leukaemia in C3Hf/PW mice
(Table W). Gross developed his potent Passage A filtrate (Gross, 19576) by
selecting an active Ale leukaemic extract and passing it through serial cell-free
inoculation of newborn C3Hf/Gs mice. In the work reported here, a potent
extract was obtained in a similar way but in a relatively shorter period of time,
the extracts being passed through successive generations of newborn Ak mice
instead of CSHf/Gs mice (Table V). No doubt this procedure must select and
concentrate active agent, since dilution of the final potent extract reduces the total
incidence and delays the average age of onset of leukaemia in inoculated mice
(Gross and Drejduss, 1959).

Subline differences exist in respect to susceptibility to leukaemogenesis by any
given extract as was pointed out by Gross (1955a) and Woolley and Small (1957).
In this study, a valid comparison can be made of the response of 2 sublines of
C3H mice, both originally obtained from Bittner, since both were injected simul-
taneously with the same filtrate. Passage A. It is obvious from Table III that
the C3Hf/Gs subline is by far the more sensitive.

Only newborn mice were susceptible to the leukaemogenic activity of filtrates
prepared from tissues of Ak mice with spontaneous leukaemia. This may be due
to ;

(1) A state of immunological unresponsiveness ;

(2) a concentration effect ;

(3) factors, other than immunological, inherent to the particular stage
of development of the animals at birth.

(1) It has been suggested that immunological tolerance is the reason why only
newborn mice axe susceptible to an agent recently isolated from a foreign strain
(Harris, 1968 ; Barnes et al., 1959). In this study, a state of acquired tolerance
in C3H mice to transplantation antigens of Ak mice did not appear to give toler-
ance to the Ak leukaemic agent (Table VIII). The mice were fully susceptible
to grafts of Ak leukaemic cells but not to the leukaemogenic activity of the Ak
agent. For the induction of tolerance, normal Ak cells, which according to Gross
(1953, 1959) contain the agent, were injected intravenously at birth, and yet no
leukaemia developed. Perhaps the quantity of agent in 5 million normal Ak
cells is not sufficient for leukaemogenesis, or perhaps the agent is intracellular
ant.genically distinct and must be introduced in the free state in newborn animals’

(2) bimple quantitative factors may serve to explain the age susceptibfiitv to
the leukaemogenic effect of cell-free filtrates. For any active filtrate the con
centration of agent present in the newborn after inoculation must be relativelv ffir
^eater than that which can be attained in an adult mo^r AugmiSon S

infect.,., ty presnmaMy occurs as a result of selection of the mostlctiv^S^acJ

J. F. A. P, MILLER

after serial passage tlirough successive generations of mice of any one strain A
far greater concentration of active agent can then be achieved foDowing inocu-
lation into suckling or young adult mice. This may explain why acceleration of
leukaemia in Ak mice by Ale filtrate can only occur when the filtrate is given at
birtli, and Avhy, on the other hand, Passage A can accelerate Ak leukaemia when
given to older Ak mice or can produce leukaemia after inoculation into voniw
adult C3Hf /Gs mice. ^ °

(3) Shubile has pointed out that the response of new'born mice to filtrates
should also be viewed as a response of a sensitive biological system to carcino-
genesis (Pietra, Spencer and Shubik, 1959). He reports a high incidence of early
malignant lymphomas in a relatively insusceptible strain of mice following the
inoculation at birth of a single low”^ dose of chemical carcinogen.

All the leukaemias developing in our inoculated mice -were lymphoid with
thymus involvement resembling typical spontaneous lymphomas in Ak mice.
They were all transplantable to mice of the recipient line, and many were trans-
plantable to both donor and recipient strains. This confirms similar observations
made by Purth et al. (1956) and Gross (1958c). No other tumours, nor tumours
characteristic of pol3’'oma virus infection (Stewart et al., 1957) were seen in any of
the mice of the present series in contrast to the results of other workers in this
field (Stewart, 1955 ; Law et al., 1955 ; Salaman, 1959). Salaman, for instance,
recorded the occurrence of 40 tumours (but no leukaemia) among 15 out of 23
C3Hf/Bi mice inoculated at birth with leukaemic filtrate. However, the filtrates
were prepared from leuJiaemias that had already been transplanted three times
by cell graft through AliR mice. Gross has stressed that as far as leukaemo-
genesis cell-free extracts is concerned, the source of material should either be
a spontaneous leukaemia or one induced with leukaemic extracts rather than a
transplanted leukaemia (Gross, 1956, 19586). Some supporting evidence for this
claim was obtained by Kassel and Rottino (1959) who showed that extracts
prepared from transplanted AKR leukaemic tissues resulted in the development
of parotid tumours, adrenal tumours, osteosarcomas and fibroinyxomas but no
leukaemia.

It "would be pure speculation to attempt, on the basis of the present results,
to identify the agent in leukaemic filtrates as a virus or as a genic complex derived
from either neoplastic or potentially neoplastic cells. An increase in leukaemia
incidence, per se, does not establish the existence of a virus-mediated mechanism.
Final identification of the agent must await further investigations of its biological
and physicochemical properties.

STOISIABY

1. Newborn or adult mice of the low-leukaemic strains C3Hf/PW, C3Hf/Gs,
GBA/H, and of the high-leukaemic strain Ak,- were inoculated intraperitoneally
with cell-free extracts prepared from tissues of Ak mice with spontaneoiK lei '-
aemia, and of C3Hf/Gs mice noth leulraemias induced by leukaemic filtrate.

Passage A. , i •

2. Ak leukaemic filtrates inoculated into newborn low-leukaemic strain
resulted in leukaemia in 11-8 per cent of 68 C3Hf/PW mice at / to 14 mont is an
7 per cent of 71 CBA/H mice at 6 to 15 months. No leulraemia occurred wlien
these filtrates were inoculated into 34 adult C3Hf/PW mice or 18 adul /

mice.

LEUKAEJrOGENESIR EY CELL-EREE FILTRATES

3. Passage A filtrate resulted in 26-7 per cent leukaemias at 7 to 10 months
when inoculated into 4:7 newborn C3Hf/P\V mice and 100 per cent leukaemia at
2 to 4 months following inoculation into 87 one- to 14-days-old G3Hf/Gs mice.
The same filtrate produced GO per cent leukaemias when inoculated into 20 one-
to 2-months-old C3Hf/Gs mice.

4. Acceleration of the onset of leukaemia to 3 to G months occurred in G8 per
cent of 75 Ak mice inoculated at birth with Ak leukaemia filtrates but in none of
18 Ak mice inoculated at 1 to 2 months of age. Passage A filtrate accelerated
leukaemia in 100 per cent of 15 Ak mice inoculated between 1 and 14 days of age.

5. Transplantation of some of the leukaemias produced in G3H mice revealed
some ambivalence ; all were transplantable to C3H mice but many were also
transplantable to mice of the original donor Ak strain.

6. Increase in potency of an Ak Icukaemic filtrate was evident after 4 serial
cell-free inoculations of newborn Ak mice. The final filtrate was associated with
the development of leukaemia at 3 to G months in GS-S per cent of 16 03Hf/Gs
mice inoculated between 1 and 5 days.

7. 1^0 leukaemia developed in 79 C3Hf/PW mice which received at birth an
intravenous injection of normal spleen and thymus cells from health}’’ young
adult Ak mice. Fifty-nine of these mice received, in addition, intraperitoneal
injections of Alv leukaemic filtrate at 4 to 8 weeks of age. All the mice in tliis
group were fully tolerant of Ak skin grafts.

8. Only lymphocjdic leukaemias or lymphoid tumours confined to the thymus
were seen in mice of the present series. No other tumours such as those described
by other workers in this field have appeared.

I wish to express my gratitude to Dr. Ludwik Gross who supplied me with liis
strain of C3H mice and ■nith mice bearing leukaemias induced Ijy Passage A fil-
trate. I also vish to thank Professeur G. Mathe of the Association Claude-
Bemard, Paris, Madame Tuffrau of the Centre de Selection des Animaux de
Laboratoire, Gif-sur-Yvette, and Dr. Edith Paterson of the Christie Hospital and
Holt Radium Institute, IManchester, -who sent me some of their AKH mice. I
am indebted to the Gaggin scholarship from the University of Queensland, Bris-
bane, Australia, and to Professors A. Haddow and P. C. Koller and Dr. R. J. G.
Harris for their interest. The investigations have been supported by grants to
the Chester Beatty Research Institute (Institute of Cancer Research : Royal
Cancer Hospital) from the Medical Research Council, the British Empire Cancer
Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna
Fuller Fund, and the National Cancer Institute of the National Institutes of
Health, U.S. Public Health Service.

REFERENCES

Baexes, D. W. H., Ford, C. E., Ilbeby, P. L. T., Joxes, K. W. axd Loutit J. F.

(1959) Ac(a Un. int. Cancr. 15, 544.

ellinghajM, R. E. axd Medawar, P. B. — (1951) J. exp. Biol., 28, 385.

j^BucEY, M., Schilling, M. G. axd Goss, M. F.— (1957) Cancer Res.
17, 809. ’

C.— (1957) Ann. N.Y. Acad. Sci., 68, 522.

TH, J., Buffett, R. F., BAXASIE^vICz-RoDEIGUEz, M. axd Upton, A C —119561
Proc. Soc. exp. Biol, N.Y., 93, 165. ' ^ ^

J. P. A. P. raLLER

Geaffi, a. — (1957) Ann. N.Y. Acad. Sci., 68, 540.

Geoss, L. — (1951) Proc. Soc. exp. Biol., N.Y., 76, 27. — (1953) Acta haemal., 10, 18.—
(1955a) Proc. Soc. exp. Biol., N.Y., 88, 64. — (19555) Ibid., 88, 362. — (1956) Acta
haemal. 15, 273. — (1957a) Ann. N.Y. Acad. Sci., 68, 501. — (19576) Proc. Soc.
exp. Biol, N.Y., 94, 767.— (1958a) Acta haemal., 19, 353.— (19586) Cancer Res.,
18, 371.— (1958c) Proc. Soc. exp. Biol, N.Y., 97, 300.— (1959) Ibid., 100, 325.

Idem AND Deeyfuss, Y. — (1959) Proc. Amer. Ass. Cancer Res., 3, 24.

Haeeis, R. J. G. — (1958) J. chron. Bis., 8, 58.

Hays, E. F. and Beck, W. S. — (1958) Cancer Res., 18, 676.

Kassel, R. and Rotting, A. — (1959) Ibid., 19, 15^

Law, L. W. — (1957) Ann. N.Y. Acad. Sci., 68, 616.

Idem, Dunn, T. B. and Boyle, P. J. — (1955) J. nat. Cancer Inst., 16, 495.

Mjllee, j. F. a. P. — (1959) Proceedings of the seventh European Congress of Haemat-
ologj', London, Acta haemal, in press. — (1960) Brit. J. Cancer, 14, 93.

PiETEA, G., Spencee, K. AND Shdbik, P.— (1959) Nature, 183, 1689.

Rudali, G., Duplan, j. F. and Lataejet, R. — (1957) Bidl. Ass.frang. Cancer, 44, 440.

Salaman, M. H. — (1959) Brit. J. Cancer, 13, 76.

ScmvAETZ, S. 0. AND SCHOOLMAN, M. H. — (1959) Blood, 14, 279.

Stewaet, S. E. — (1955) J. nat. Cancer Inst., 15, 1391.

Idem, Eddy, B. E., Gochenoue, A. M., Boegese, N. and Gedbbs, G. E. — (1957)
Virology, 3, 380.

Woolley, G. W and Small, M. C. — (1956) Cancer, 9, 1102. — (1957) Ann. N.Y. Acad.
Sci., 68, 533.

STUDIES ON MOUSE LEUICAEMIA. THE ROLE OE THE THYMUS
IN LEUKAEMOGENESIS BY CELL-FREE LEUICAEMIC FILTRATES

J. F. A. P. illLLER

From the Chester Beatty Research Institute, Institute of Cancer Research :
Royal Cancer Hospital, Fulham Road, London, (S.ir.3

Received for publication December 11, 1959

Total thymectomy markedly reduces the high incidence of spontaneous
lymphomas in certain strains of mice (McEndy, Boon and Furth, 1944 ; Law and
Miller, 1950a). A similar effect is seen in mice in which the disease can be induced
by ionizing radiations (Kaplan, 1950), carcinogenic hydrocarbons (Law and
Miller, 19505) or the inoculation at birth of cell-free leukaemic extracts (Gross,
1959 ; Levinthal, Buffett and Furth, 1959 ; Miller, 1959a, 1959c). Thymectomy
has no effect on radiation-induced myeloid leukaemia in RF mice (Upton et al.,
1958) suggesting that the leukaemogenic influence of the thymus is specific for
lymphoid tissues. There are no differences between thymectomized and control
groups of mice in weight curves, breeding behaviour or susceptibility to common
laboratory infections. Reduction of leukaemia incidence by tltymectomy does
not seem, therefore, to be related to other factors affecting the general health of
the animals (Law and Miller, 1950a, 19506).

Subcutaneous grafts of autologous or isologous thymus in thymectomized mice
restore the potentiality of developing the disease, whether this be spontaneous,
induced by carcinogen (Law and Miller, 1950a, 19506) or by irradiation (Kaplan
et al., 1956).

The work to be reported here shows that while thymectomy prevents the de-
velopment of the disease following inoculation of leukaemic filtrate, thymus grafting
as late as 6 months after thymectomy restores the potentiality for leukaemia
development in mice inoculated at birth with cell-free filtrates. A preliminary
account of this work has been given elsewhere (Miller, 19596).

MATERIALS AND METHODS

The strains C3Hf/PW, C3Hf/Gs, CBA/H and Akj (Miller, 1960) were used.
Filtrates were prepared in the same way and from the same sources as described
elsewhere (Miller, 1960). The route of inocualtion and the doses given were also
the same.

Thymectomy was usually performed at 4 weeks of age. The thymus was
removed by suction through an incision in the neck and thoracic wall extending to
the level of the second rib. Thymus grafting was performed by introducing one
whole thymus with a trocar and cannula into the subcutaneous tissues of the right
or left axilla. Donor and recipient mice were always of the same sex and the aee
of the donor varied from 1 to 30 days according to the experiment. Strict asepsis
was observed during the grafting procedure. ^

J. F. A. P. MILLEE

EXPERIMENTAL

Tliree experiments were set up as follows :

Experiment I. — Mice of all 4 strains were inoculated at birth with a filtrate of
leultaemic tissue. At 4 weeks of age about half the inoculated mice were tlij^mec-
tomized. Some of the thymectomized mice received a further inoculation of
leukaemic filtrate after thymectomy.

Experiment II. — ^Normal mice of the G3Hf/Gs strain were thymectomized at
about 4 weeks of age and received from 1 day to 1 month after thjTOectomy a
subcutaneous thymus graft. The thjonuses were obtained either from normal
newborn C3Hf/Gs mice (Group I) or from C3Hf/Gs mice betv^een 5 and 30 days
of age that had themselves been inoculated at birth with Passage A filtrate
(Group II).

Experiment III. — Two groups of mice were studied : the first was inoculated
with leukaemic filtrates soon after birth and thymectomized 4 weeks later, and
the second was first thymectomized at about 4 weeks and inoculated with leukaemic
filtrate just after thymectomy. The mice in both groups were grafted sub-
cutaneously with day-old thymuses from normal C3Hf/Gs mice. These grafts
were performed from 1 day to 6 months after thymectomj’^ in the first group, and
from 1 to 2 months after thymectomy in the second group.

RESULTS

Experiment I. — The development of lymphocytic le^Ikaemia in thymectomized mice
inoculated at biiih with levlkaemic filtrates (Table I)

Thymectomy prevented leukaemia in all but 3 Akj mice of the strains tested.
This was particularly striking in the C3Hf/Gs strain where Passage A inoculation
into mice at or soon after birth was followed by 100 per cent leukaemias in non-
thymectomized littermates. Further inoculation of extracts after thymectom}'
did not raise the incidence of leukaemia in thymectomized hosts. The incidence

Table I. — Experiment I. — Incidence of Lymphocytic Leulcaemia
in Thymectomized Mice

Mice with

IjTnphocytic leukaemia*

strain

Filtrate given
at birth

ThjTnus

Number
in group

Number

Age

in months

Per cent

C3Hf/PW .

Ak leukaemic

r Intact

113

7-14

17-7

or Passage A

\Kemovedt

Passage A

/Intact

2-4

100

C3Hf/Gs

\Eemovedt

• —

Ak leukaemic

/Intact

6-15

CBA/H

"pEemoved

■

/Intact

3-6 1

78-2

Aki

Ak leukaemic

{

/Removed

9-10 /

3, 6 and 10

4-S

* Survivors are over 12 months of age. r i

t Some of these mice received further inoculations of leukaemic filtra e

after thj’mectomy.

KOl.K OV THYMUS IN l.HUKAKMOGKNESIS

of spontaneous leukaemia in untreated mice of tlie strains used hero lias been
reported elsewhere (Miller, 19G0).

Experimeni II. — The. derdopment of b/mphoid tumours in iJn/muscs grriffcd to
tlujmcctomizcd mice (Table II)

The fate of thymuses grafted to normal hosts was examined. Thymuses taken
from normal donors did not develoji lymjihoid tumours (Grouji I). On the other
hand, thj-muses taken from donors which had themselves been inoculated with
leukaemic extracts at birth became malignant in some of the uninoculated hosts
three to five months after grafting (Group II).

Table II. — Experiment II. — Incidence of Lijmphoid Tumours in Thymuses
Grafted to Thymcctomizcd ClillfIGs Mice
Donor C3Hf/Gs*

Host C3Hf/Gst

. A

-Age (in

r — —

-A. ^

days) of

Age of

Mice With lymphoid tumours

Treatment

thymus

Number

host at

given at

when

of mice

grafting

Age

birth

grafted

graftetl

(months)

Number in months

Per cent

None

1-2

0 —

Passage A

r 5-9
\ 10-30

. 3 5-7

10 .5-0

58'4

* Donors in Group I were not inoculntcd. Donors in Group II received Pnssogo A iiltrnto nt
birth.

t Hosts in either group were not inoculntcd with filtrates. They were nil thymeotomized nt
1 month of age.

Experiment III. — The development of lymphoid tumours in normed thymuses grafted
to thymectomized hosts inoculated with leukaemic filtrate (Table III)

The fate of thjTiiuses from normal donors was studied in inoculated thj’^mec-
tomized hosts. About half these thymuses developed lymphoid tumours when
introduced as late as 6 months after th3’^mectomy and almost all became malignant
when grafted within three months after thj^mectomj'^ (Group I). Lymphoid
tumours rvere diagnosed from 2 to 4 months after grafting.

ThjTuuses introduced into hosts inoculated after thjTOectomy also developed
Ijnnphoid tumours, but only in one-third of the mice (Group II).

Table III. — Experiment III. — Incidence of Lymphoid Tzimours in Thymuses
Grafted to CZHfjGs Mice Inoculated ivith Leukaemic Filtrates

Mice with lymphoid tumours

Age of hosts
Filtrate when grafted
Strain given (months)t

Group

C3Hf/Gs

Passage A

2- 3

3- 4

2-3

Number

mice

Number

Age

in months
3-4
3—5
5-8
9-11

7-8

'1^ Group I were inoculated at birth and later thrunectomized. Mice in Groun IT
i ectomized at 24 days and inoculated between 35 and 40 days

Per cent
100
83-3
75

54-5

33-3

uniLtlted hialthrLt'^us^^^^^^^^ thjnnuses from

J. F. A. T. MILLEB

Transplantation

transplanted in all cases to untreated 1- to 2-iuontIis

old o3Hf/Gs mice.

In the majority of cases, the tumours in Experiments II and III appeared at
tost to be confined to the subcutaneous spaces where the thymus had been grafted
At tins stage the spleens from these animals did not produce leukaemia after
transplantation. Later, dissemination took place and generalized leukaemia
became evident in the thymus grafted hosts. These transplantation studies are
sfcjiJ in progress.

DISCUSSION

Our results confirm the previous findings that thymectomy prevents the
development of lymphomas in mice inoculated with leukaemic filtrates (Gross,
1959 ; Levinthal et al., 1939 ; illiller, 1959a). They show, in addition, that the
potentiality for leukaemia development is still present in the inoculated thymec-
tomized host, and that normal thymuses can express tliis potentiality when grafted
to a subcutaneous site in the inoculated thymectomized host as late as 6 montlis
after thymectomy.

Thymectomy might prevent the development of leukaemia following inocula-
tion of leukaemic filtrates by effecting removal of :

(1) The source of the leukaemic agent ;

(2) the site of multiplication of the agent ;

(3) the cells most susceptible to leukaemic transformation ;

(4) the source of a humoral factor involved in leukaemogenesis.

(1) In the present experiments, normal thymuses grafted in thymectomized
inoculated hosts as late as 6 months after thymectomy developed Ij'mphoid
tumours. It carmot be maintained, therefore, that thymectomj'- prevents leuk-
aemia development in inoculated mice by removing either the source of the
leukaemic agent or the site where the agent is principally stored. Tliis does not
exclude the possibility that the thymus may contain some of the agent in inoculated
mice (Gross, 1959) but it does exclude the suggestion that the thymus is the onlj'
source of the agent. It follows that the agent might be recoverable from tissues
of inoculated hosts up to 6 or more months after thymectomy. Experiments are
now in progress to determine whether this can be done.

(2) It is possible that the leukaemic agent must reach a critical concentration to
produce leukaemia in its host. If so, the fact that no lymphoid tumour occurred
in inoculated thymectomized mice until a thymus graft was introduced can be
interpreted to mean that the agent multiplies satisfactorily only in thymus tissue.

(3 } In every case of leukaemia following the inoculation of leukaemic nitrates
the thymus was involved, and in some cases it was the sole organ involved. le
results obtained in Experiment II show that cells capable of leukaemic trans or
mation are present in the thymus as early as 5 to 10 days after the inocula jon o
leukaemic filtrates. Removal of such a potentially malignant focim won ms
prevent the development of the disease. Thymus involvement in G58 imce ana
in DBA/2 mice painted with methylcholanthrene is, however, very rare ( av an
hliller, 1950a, 19505) and yet the disease can be prevented by thymectomy.

BOLE OF THYJMUS IN LEUKAEMOGENESIS J ‘

is difficult to assume that such a procedure, in this case, simply acts by removing
the cells most susceptible to leukaemic transfoi’ma,tion.

The evidence obtained from the present work is not sufficient to decide for or
against any or both of these last two possible explanations. The fact that thymus
grafted to inoculated thymectomized hosts develops lymphoid tumours can be
taken as supporting evidence for either of these hypotheses.

(4) Our results do not exclude the possibility that a humoral factor might be
involved in leukaemogenesis in hosts that are conditioned either by irradiation,
or chemical carcmogens, or the inoculation of leukaemic filtrates. The possibility
of a non-cellular factor from the thymus exerting an influence in the leukaemo-
genic process has been stressed by Law (Law and kliller, 1950a ; Law and Potter,
1956), 'and Metcalf (1958), who suggests that tliis factor is liis thymic lymphocy-
tosis Simulating factor Avhich he has shown to stimulate lymphocyte proliferation
in the mouse.

sumiABY

1. The effect of thymectomy and thymus grafting on the leukaemogenic
activity of cell-free leukaemic extracts has been investigated.

2. No leukaemia occurred in 59 C3Hf/PW, 38 C3Hf/Gs and 28 CBA/H mice
inoculated at birth Avith leukaemic filtrates and thymectomized at 4 weeks of age.
The incidence of leukaemia in non-thymectomized inoculated mice of the same
strains was 17-7 per cent of 113 C3Hf/PW mice, 100 per cent of 45 C3Hf/Gs mice
and 7 per cent of 71 OBA/H mice.

3. Only 3 leukaemias occurred in a group of 62 thymectomized Akj mice Avhich
were inoculated at birth with leukaemic filtrates. The incidence of the disease in
69 non-thymectomized inoculated Aki mice Avas 78-2 per cent the majority of the
mice succumbing between 3 and 6 months of age.

4. Thymuses from normal day-old C3Hf/Gs mice grafted to normal adult
thymectomized C3Hf/Gs mice did not develop lymphoid tumours or induce
leukaemia in their hosts. Prom 30 to 60 per cent of thymuses from 5 to 30 days
old C3B[f/Gs mice inoculated at birth with leukaemic filtrate (Passage A) deve-
loped lymphoid tumours Avhen grafted to normal uninoculated thymectomized
C3Hf/Gs mice.

5. Thymuses from normal, uninoculated, day-old, C3Hf/Gs mice Avere grafted
to adult thymectomized C3Hf/Gs mice which had themselves been inoculated at
birth AAuth Passage A filtrate. Prom 50 to 100 per cent of these thymuses deve-
loped lymphoid tumours in inoculated mice grafted from 1 day to as late as 6
months after thymectomy.

6. The implications of these results are discussed. It is concluded that the
potentiality for leukaemia development is still present in inoculated thymecto-
imzed hosts for many months after thymectomy and that thymus grafting at any
time Avill express this potentiality in full.

I am indebted, to the Gaggin scholarship from the University of Queensland
llrisbane, Australia, and to Professors A. Haddow and P. C. Koller and Dr R J
Harris for their interest. The investigations have been supported by grants to
tlie Chester Beatty Research Institute (Institute of Cancer ResearchT Royal
Cancer Hospital) from the Medical Research Council, the British Empire Cancer
Campaign, the Jane Coffin Childs Memorial Fund for Medical Research! the Anna
9

J. F. A. P. MILLER

Fuller Fund, and the National Cancer Institute of the National Institutes of

Health, U.S. Public Health Service.

REFERENCES

Gross, L. — (1959) Proc. Soc. exp. Biol., N.F., 100, 325.

ICaplan, H. S. — (1950) J. nat. Cancer Inst., 11, 83.

Idem, Carnes, W. H., Brow, M. B. and Hirsch, B. B. — (1956) Cancer Res., 16, 422.

Law, L. W. and JIiller, J. H. — (1950a) J. nat. Cancer Inst., 11, 253. — (19506) Ibid.,
11, 425.

Idem AND Potter, M. — (1956) Proc. nat. Acad. Sci., 42, 160.

Leatnthal, j. D., Buffett, R. F. and Forth, J. — (1959) Proc. Soc. exp. Biol., N.Y.,
100, 610.

McEndy, D. P., Boon, M. C. and Forth, J. — (1944) Cancer Res., 4, 377.

Metoat.f, D. — (1958) Ann. N.Y. Acad. Sci., 73, 113.

JIiLLER, J. F. A. P.— (1959a) Nahire, 183, 1069.— (19596) Ibid., 184, 1809.— (1959c)
ftoceedings of the seventh European Congress of Haematology, London, Acta
haemat., in press. — (1960) Brit. J. Cancer, 14, 83.

Upton, A. C., Wolff, F. F., Forth, J. and Kemball, A. W. — (1958) Cancer Res., 18,
842.

STUDIES OE ELUID INIEDIA FOR THE CULTIVATION OF MOUSE
ASCITES TTOIOUR CELLS IN VITRO

A. K. POWELL

From the Department of Experimental Pathology, Mount Vernon Hospital,

Nortlnvood, Middlesex

Keceived for publication Januarj- 28, 19G0

Ascites tumours in experimental animals are essentially almost pure popu-
lations of suspended tumonr cells multiplying in peritoneal exudate. Amoebo-
cytes and shed peritoneal cells are usually present in relatively low numbers. The
suitability of these tumours for quantitative studies on malignant cells has long
been realised. As a measure of chemotherapeutic activity, Lettre (1941, 1950)
used differences in the survival times of control and treated mice injected udth
uniform volumes of ascites tumour fluid and reported that increases in body
weight of inoculated mice were proportional to increases in numbers of the tumour
cells. This latter relation was shown by Klein (1951) to be valid only for inocu-
lations vdtli certain numbers of tumour cells.

The value of Ehrlich ascites tumour cells for quantitative studies on growth
and biochemistry has been discussed in detail by Klein (1950, 1951). These
particular advantages include ease of serial sampling, low incidence of necrotic
cells, uniform nutritive conditions and distribution of cells in the exudate, and
direct action of agents on the tumour cells. In vitro cultivation of ascites tumour
cells in fluid media offers additional advantages. It affords more precise control
of the extracellular medium and environment, and direct microscopic observation
of the tumour cells in situ. A fluid medium supporting multiplication of Ehrlich
ascites tumour cells in vitro has therefore been devised.

5IATERIALS AKD ^METHODS

Mice and tumours. — Ehrlich ascites tumours were maintained in 12- to 15-
week-old RIII strain mice. They were propagated by serial intraperitoneal in-
jections of 0- 1-0-2 ml. volumes of ascites tumour fluid taken from mice inoculated
6 to 7 days previously. The 10 to 12 mice in each experimental group were
inoculated vdth ascitic fluid from one donor. The mi ce were given normal lahor-
atorj' diet and water ad libitum.

Tissue culture . — Ascites tumour fluid Avas withdraivn asepticaUy from mice
inoculated 6 to 7 days preAUously with tumour cells and killed by dislocation of
the neck. Pooled ascitic fluid from the mice of a single inoculation group was
used for each indiAudual experiment. A portion of this pooled ascitic fluid was
reserved untreated for addition as cell inoculum to the culture medium The
remainder was centrifuged for 10 minutes at about 3000 r.p.m. in a bench centri-
iuge, tlie supernatant “ascitic plasma” remoAmd and stored at 4° for later use In
the present context “ascitic fluid” refers to the ceU-containing peritoneal exudate

100

A. K. POWELL

as removed from mice, and “ascitic plasma” to cell-free supernatant obtained bv
centrifugation. ^

Earle’s buffered saline solution (Earle, 1943) was sterilised hy Seitz filtration
and adjusted to pH 7-4-7-6. It rvas used as the solvent for trj^sin and hyalu
ronidase. Trypsin (B.D.H., commercial grade) was made up at a concentration
of 1 mg. per ml. and sterilised by Seitz filtration. Hyaluronidase (“Rondase”
Evans) was dissolved in sterile saline to give a concentration of O-o mg. per ml'
Both enzyme solutions were used at pH 7-4-7-6 and prepared freshly for use.
Heparin (“Liquemin”, Roche) was prepared in 0-85 per cent sodium chloride
solution at a concentration of 25 i.u. per ml. It was ampouled in convenient
volumes, autoclaved and stored at 4°.

Embrjm extract was prepared from mouse embryos of the RIII strain taken
at about the 15th day of development. The freed embryos were cut into large
fragments, blood removed by repeated washings with Earle’s solution and the
tissue then finely minced with curved scissors to give an almost homogeneous pulp.
This was churned vdth Earle’s solution by means of a coarse pipette and stood at
laboratory temperature for 2 to 3 hours before being centrifuged for 5 minutes at
3000 r.p.m. The ratio of tissue to supernatant after centrifugation was appro.ff-
mately 1:2.

In vivo ascites tumour fluid is churned to homogeneity by peristaltic and body
movements. In this way fresh nutrients are supplied to the tumom- cells and
their waste products do not accumulate locally. The churning was simulated by
cultivating the ascites cells in standard hexagonal roller-tubes which were revolved
in a drum at 11 r.p.m. Each tube was loaded with 2-5 ml. of medium. The
medium variants were replicated in each experiment. With the exception of the
heparin and Earle’s solution, all components of the medium, including ascitic fluid,
were prepared as soon as possible before use.

After incubation for 24 hours in suitable media the tumoiu cells tended to be
attached loosely to the surface of the roller-tubes, especiall}'^ adjacent to the angles
between the lateral faces. However, they were readily detached by rotation of
the tubes between the palms and a suspension of mostlj' discrete tumour cells was
obtained. Macrophages tended to adliere firmly to the glass but a method of
virtually complete recovery of all cells was found. The roller-tubes were coated
successively with a silicone (“Repelcote”, Hopkins and Williams) and 2 per cent
paraffin oil in ethyl ether. The tubes were drained and the ether allowed to eva-
porate. They were then dry-sterilised. Chilhng the culture tubes in ice-water,
followed by shaking, detached macrophages. Samples for examination were
taken from the homogeneous cell suspensions. Dilute saline solutions of crude
trypsin were not completely effective for recovery of macrophages from uncoated
roller-tubes although the tumour cells themselves w^ere completely dispersed.

BXPERIiMENTAL RESULTS

The experiments refer to Elurlich ascites rumour cells unless otherwise stated.

Aceto-orcein preparations w'ere used to assess the incidence of mitosis an
condition of the cultivated cells in the preliminary experiments. Since asci ic
plasma itself would be expected to contain all the factors necessar}'^ for the grou j
of the tumour cells it ivas tested as such. After incubation of w'hole unmoc i e
ascitic fluid for 24 hours no dividing cells were observed and almost all the urao

CULTIVATION OT jSIOUSE ASCITES TUMOUR CELLS

101

cells were dead and embedded in fibrin clots. The macrophages present survived
far better than the carcinoma cells. The tumour cell population of ascitic fluid
in vivo during the logarithmic phase of ^owth may be assumed to be near the
maximum allowed by the gi’owth-supporting properties of the peritoneal exudate.
Warburg and Hiepler (19.52) have demonstrated the nutritive poverty of ascites
tumour fluid. Because of this and the absence in in vitro conditions of renewal
of nutrients and removal of waste products bj’^ the host, population densities in
vitro equivalent to those in vivo would be expected to exhaust rapidly the medium.

Accordingly a series of experiments was made in which the proportion of tumour
cells per unit volume of the media, wliich consisted of ascitie fluid and plasma,
whole or diluted wflth Earle’s solution, w'as varied. In view of the possible im-
portance of fibrinogen to the tumour cells ascitic plasma containing 1/lOth by
volume of heparin solution was used as a diluent instead of ascites tumour serum.
These media did not support proliferation although in some the great majority of
the carcinoma cells remained viable after incubation for 24 hours. Similar media
supplemented with embrjm extract to provide growth-stimulating factors gave
improved but still unsatisfactory results. Further experiments led to the in-
clusion of trypsin and hyaluronidase in the culture media.

In the definitive experiments the media consisted of heparin, trypsin and
hyaluronidase solutions, embryo extract, ascitic plasma and ascitic fluid, combined
in this order to avoid formation of fibrin and possible injury to the tumour cells.
At each stage the pooled components were thoroughly mixed before the addition
of the next component.

Media with varying proportions of ascitic plasma to standard amounts of the
other components were tested. It was established that the optimum proportion
of ascites plasma, including the ascitic fluid as plasma, was about 40 per cent of
the total medium to 60 per cent for the combined saline components. Native
horse serum was substituted for ascites plasma in some experiments and found to
maintain the tumour cells in good condition but not to promote cell division.

An empirical medium of ascitic plasma 2 parts by volume, embryo extract 2
parts, heparin, trypsin and hyaluronidase each 1 part, and ascitic fluid 1 part, was
adopted as a basis for further study. The plasma moiety, including ascitic fluid,
was 37| per cent and the total saline components 62| per cent by volume. The
packed cell volume of ascitic fluid was about 33 per cent in most instances.

To determine the relative importance of the indimdual components the basic
medium was modified by the substitution of Earle’s solution for each saline

component, including heparin, separately. The effect of increased numbers of
tumour cells inoculated into the medium was also determined. The media are
listed in Table I and the results of a typical experiment in Table II. The cell
population of the ascitic fluid used as inoculum and of the various media after
cultivation for 24 hours were determined by Neubauer haemocytometers, using
white cell pipettes. Two or more separate preparations were made for each
medium. The media were diluted for counting with 0-05 per cent eosin in Earle’s
solution. Dilute eosin solutions have been used to estimate cell viability bv
Schrek (1936), I^em (1951), and Klein and Revesz (1953). Staining of ceuLal
be taken as an indication of damage but not aU stained ceUs are non-viable (British
Empire Cancer Campaign, 1951). Unstained cells are, however, viable

iJie results of a typical experiment are given in Table II. In this narticular
trial the population of the parent ascitic fluid was found to be 105, 750 ceUs per

S.M.S. MEDICAL COLLEGE,

tlTWR A "R.*^ * 7 O ^ 5^ X A T-r^ ri ft.

102

A. K. POWELL

'^—Composition, Given in Aliquots by Vohime, of Experimental Media
Differing in Absence of Single Components from the Basic Medium [Eo. 1 )

Abbreviations used : ]\IX — embrj^o extract ; HP — heparin ; HY

hyaluronidase ; TR — ^trypsin ; AP — ascitic fluid.

Components of media
Ascitic plasma
Embrj'o extract
Heparin solution
Hyaluronidase solution .
Ti^^psin solution
Ascitic fluid .

Earle’s solution

Media numbers

t ~

1 2

2 2

2 —
1 1

1 1

I 1

1 1

3 4
2 2
2 2
- 1
1 —
1 1
I 1
1 1

5 G

2 1

2 2

1 1

1 2

1 —

Component tested .

MX HP HY TR AE

mm.3 and that of each medium except No. 6, was calculated to be 13,219 mm.^
The incidence of eosin-stained cells was verj' nearly 1 per cent. Differential
counts of tumour and non-tumour cells were not made because of the low incidence
of the latter and the inherent margin of error in haematoc 3 d;e determinations.

The amount of fibrinogen and consequent extent of clotting varied with indi-
vidual samples of ascitic fluid from different mice. In this instance the medium
without heparin clotted only slightly and supported increased growth. Heparin
appeared to be inhibitory at the concentration used in the basic medium but it is
possible that the replacement of sodium chloride by balanced saline solution had
some effect. Heparin has been shown bj"^ HeObrumi and Wilson (1949) to inhibit
cell division and to affect the viscosity of protoplasm. Embryo extract, h 3 ’alu-
ronidase and trypsin were necessary for appreciable cell multiplication but the
incidence of eosin-stained cells in the respective cultures was significantly in-
creased, compared with the basic medium, only in the absence of embryo extract.
Doubling the initial population of tumour cells had a definite effect on proliferation.
This was possibty due to increased amounts of soluble protective factors liberated

Table II. — Effects on Cell Population of Absence of Individual Components
of the Basic Medium after Incubation for 24 hours

^Medium

No.

Component

tested

(Basic Medium)
Embryo extract
Heparin
Hyaluronidase
Trj^psin
(Ascitic fluid)

Initial

population

(mm.’)

13,219

26,438

Population
after 24 hours
(mm.’)
16,250
11,700
18,800
13,925
12,500
43,300

Percentage
difference
after 24 hours
-}-23
-11-4
-f-42-2
-i-5-3
-5-4
-(-G3-7

Percentage
of eosin-
stained cells
after 24 hours
3-3
11-9
7-4

3- 8

4- 8
29-0

by the cells into the medium. The significance of these factors has been discussed
previously (Powell, 1957). Apart from the medium lacldng embryo extract
increased frequencies of damaged cells were associated with high proliferatioi

rates and resulting exhaustion of media (No. 3 and 6).

The efifects of density of population were further investigated by var 3 ing
proportions of ascitic fluid to ascitic plasma but raamtaimng the
of these at 37 1 per cent of the complete basic medium. The results of a t 3 pical

CULTWATION 01? MOUSE ASCITES TUMOUR CELLS

103

experiment are given in Table III and illustrate the characteristic relation between
density of population and cell multiplication and degeneration, respectively. The
incidence of eosin-stained cells was also related to the increase in population
during the incubation period.

Table 111— Effects of Increasing Initial Cell Populations
upon Proliferation in Basic Medium

The two higher initial populations were calculated on the basis of that deter-
mined for the lowest.

Initial

population

17,614

35,228

52,842

Population
after 24 hours

23,850

35,000

47,350

Percentngo difference
in population
after 24 hours
-I-35-9
±

-10-3

Percentage of
eosin-stained colls
after 24 hours
2-9
1-8
24-4

Initial population densities significantly less than l/8th of the in vivo density
were found to be unfavourable for rapid multiplication. This was attributed to
the inability of the cells in these numbers to produce an adequate concentration
of diffusible protective factors in the medium. In general it appeared that in
2-5 ml. of the basic medium a tumour cell population of about 1 /8th of the normal
range in native ascitic fluid gave a convenient compromise between cell prolifer-
ation and degeneration during an incubation period of 24 hours.

Attempts were made to cultivate Sarcoma 37 ascites tumour cells in similar
basic medium with homologous ascitic plasma. These attempts failed. How-
ever, multiplication rates comparable to those found in vivo were obtained with
a modified medium in which ascitic fluid constituted l/8th of the total volume.
In this one of the two parts of the embryo extract was replaced with one of tumour
cell extract; in all other respects the basic medium was unchanged. To prepare
this extract the sedimented cells of centrifuged Sarcoma 37 ascitic fluid Avere
suspended in an amount of Earle’s solution equal in volume to that of the super-
natant plasma removed and the suspension incubated for 3 hours at 37°. The
cell suspension Avas shaken at intervals during the incubation, finally centrifuged
and the supernatant used as extract.

The tumour cell extract presumably contained ample essential solutes not
sufficiently provided in the unmodified basic medium. The practical limitation
of the latter set by the necessity of using a relatively high initial population of
tumour cells to obtain adequate multiplication and the consequent depletion of
the medium within 24 hours was overcome by the inclusion of the tumour cell
extract in the basic medium. Sarcoma 37 tumour cells greAv in this supplemented
medium for several days when initial population densities 1/lOOth of that of the
native ascitic fluid were used. Ehrlich carcinoma ascites tumour cells behaved
similarly in supplemented homologous media in Avhich the soluble factors Avere
supplied by the cell extract.

DISCUSSION

Tlie media described Avere able to support the groAvth in vitro of the ascites
tumour cells tested. They had the disadvantage that two of the components
Avere prepared from native ascites tumour fluid. No suitable substitutes for ascitic
plasma and serum or tumour cell extract Avere found. The latter was not
entirely replaceable by embryo extract.

104

A. K. PO'VVELL

BlirJich carcinoma and Sarcoma 37 ascites tumour cells differed qiiantitativelv
m them dependence on tiie concentration in the culture medium of the soluble
essential substances released by the cells and supplied in the cell extract The
ascitic plasma used presumably eontamed a low proportion of these substances
since Ehrlich carcinoma cells grew at certain population densities uathout the
addition of cell extract. The importance of these soluble factors for the viability
and groudh of ascites tumour cells has been discussed previously (Povell, 1957)
The present researches confirmed this earlier work. The limiting factors in the
dependence of the tumour cells upon these substances may be the ratios between
the rates at which they are synthesised and exchanged between the cells and
medium. The tumour cell extract component of the media ma5'^ be more important
for the growth of the tumour cells than the plasma fraction.

The media described would appear to be suitable for quantitative studies on
the effects of cytotoxic agents on tumour cell populations under controlled con-
ditions in vitro. Relatively large initial populations of tumour cells, 1 /8th of the
in vivo density, could be studied for periods of 24 hours or smaller initial popida-
tions for longer periods. Cultivated tumour cells inoculated intra-peritoneally
into mice gave rise to normal ascites tumours.

The roles of the enzymes used in the basic medium are uncertam. Trj'psin
has been used frequently to dissociate the cells of tissues; Moscona (1952), and
Moscona and Moscona (1952) used it upon chick embryo tissues. Willmer (1945,
1954) has described its use for this purpose and also its effect in causmg fibrocides
to round up with retention of viability. Tliis latter property may be associated
with its beneficial effects in the medium. On the other hand, it is improbable
that the enzyme remained active for long periods in the presence of the con-
siderable amounts of protein in the medium. It is possible that its proteolytic
effect upon the ascitic plasma was important shice carcinoma cells liberate pro-
teolytic enzymes and the ascites cells w'ere cultivated at densities of population
lower than those in vivo. The enzyme perhaps compensated for the reduced
numbers of cells in tliis respect and may harm liberated nutrients form the plasma
proteins.

Hyaluronidase, wliich depolymerises hyaluronic acid dermatives, reduces car-
tilaginous matrix in vitro (Paff and Seifter, 1950) and dissolves intercellular cement-
ing substance. Possibly it affected the surfaces of the ascites cells. Embryo
extract presumably functioned in virtue of its content of growth substances.

During the past decade many’^ reports of successful cultivation of cell suspended
in fluid media have been made (Paul, 1959). These include L strain fibroblasts
(Earle et al, 1954, 1956 ; Danes, 1957 ; McLimans et al, 1957), HeLa human car-
cinoma cells (Gey, Bang and Gey, 1954; Earle et al, 1956). In most of these
instances the suspended cells haA^e been subjected to violent continuous a^tation
and standard media have been used. A substrain of de Brujm’s MB lymphoblast
has been gromi in slow suspension by Owens, Gey and Gey (1953) but these cells
are atypical and altered from the parent strain.

The present researches differ from these examples of true suspension cultures
in that the cells lay mainly on the surfaces of the culture vessels although smooth y
contoured dividing cells were found free in the medium and suspensions of cells
were easily recovered from the roller-tubes. As with the lymphoblast cells of de
Bruyn the present results were probably due to the innate properties of the ascites
tumour cells reinforced by a medium consisting largely of their normal pabulum.

CULTI\’'ATION OF MOUSE ASCITES TUMOUR CELLS

105

SUMMARY

A fluid culture medium which supports the groudh of Ehrlich carcinoma
ascites tumour cells in vitro is described.

This medium is suitable for short term assays of the effects of cytotoxic agents
on the cultivated tumour cells, since they can be quantitatively recovered for

enumeration. .

The numbers of cells inoculated into this medium must lie within narrow limits

for successful cultivation.

Supplementation of the basic medium ndth a saline extract of homologous
ascites tumour cells permits successful cultivation with much smaller initial num-
bers of cells. Sarcoma 37 ascites tumour cells, which fail to grow in the basic
medium, multiply in supplemented medium.

This saline extract is presumed to contain soluble protective factors which
diffuse from the cells into the medium. The concentration of these substances
determines the number of cells which may be successfully inoculated in cultures.

I am indebted for assistance with the in vitro researches to IMr. G. A. Butcher
and with the maintenance of the tumours in vivo to ^Ir. E. Butcher.

The expenses of this work were defrayed from a block grant by the British
Empire Cancer Campaign.

REFERENCES

Beitish Empire Cancer Campaign. — (1951) Ann. Rep., 29, 57.

Danes, B. S. — (1957) Exp. Cell. Res., 12, 169.

Earle, W. R. — (1943) J. nat. Cancer Inst., 4, 165.

Idem, Bryant, J. C., Schtleing, E. L. and Evans, V. J. — (1956) Ann. N.Y. Acad. Sci.,
63, 666.

Idem, Schilling, E. L., Bryant, J, C. and Evans, V. J. — (1954) J. net. Cancer Inst., 14,
1159.

Gey, G. 0., Bang, F. B. and Gey, M. K.— (1954) Tex. Rep. Biol. Med., 12, 805.
Heilbrunn, L. Y. and Wilson, W. L. — (1949) Proc. Soc. exp. Biol., N.Y., 70, 179.
Klein, G. — (1950) Cancer, 3, 1052. — (1951) ‘ The Production of Ascites Tumours in
JDce and Their Use in Studies on Some Biological and Chemical Characteristics
of Neoplastic Cells ’. Uppsala (Almqvist and Wicksells Botryckeri).

Idem AND Revesz, L.— (1953) J. nat. Cancer Inst., 14, 229.

Lettre, H.— (1941) Z. physiol. Chem., 268, 59 ; 271, 192.— (1950) Z. Krebsforsch., 57, 1.
McLimans, W. F., Giardinello, F. E., Daaus, E. V., Kucera, C. J. and Rake, G. W.

— (1957) J. Bact., 74, 768.

Moscona, a.— (1952) Exp. Cell Res. 3, 535.

Moscona, H. and Moscona, A.— (1952) J. Anal., 86, 287.

Owens 0. von H., Gey, G. 0. and Gey, M. K.— (1953) Proc. Amer. Ass. Cancer Res
1, 41. ■’

Pafp, G. H. and Seifter, J.— (1950) Anat. Rec., 106, 525.

Paul, J. (1959) Cell and Tissue Culture ’. London (Livingston)

Powell, A. K.— (1957) Brit. J. Cancer, 11, 274, 280, 478
Schrek, R.— (1936) Amer . ./. Cancer, 28, 389.

Warburg, 0. and Heipler, E.— (1952) Z. Naturf., 7b, 193.

Yillmeb, E. N.— (1945) ‘ Grovdh and Form in Tissue Cultures ’. In ‘Growth and Form
Essays presented to D^rcy Thompson’, ed. W. Le Gros Clark and P. B. Medawar

‘T™ Culture', 2ud edition. London

106

URINARY /?-ULUCURONIDASE ACTIVITY IN CANCER OF THF
BLADDER AND OTHER DISEASES

F. J. W. LEWIS ^ND CONSTANCE H. J. PLAICE
From the Department of Pathology, Southmead Hospital, Westbury-on-Trym, Bristol

Keceived for publication Januarj- 3, 1960

A THEORY of the mode of action of aromatic amines in producing carcinoma of
the bladder in man has been developed by Boyland and his colleagues (Boyland,
Wallace and Williams, 1955; Allen, Bo3dand, Dukes, Horning and Watson, 1957;
Wallace, 1959). They pointed out that in men working in the chemical industry
who have had contact vdth a- or /?-naphthylamine or benzidine the incidence of
cancer of the bladder is very high, but other organs are not affected. They
suggest that the absorbed amines are carried to the liver where thej”^ are meta-
bolised to or/7m-aminophenols which are very rapidly”^ conjugated with sulphate or
glucuronic acid by similar mechanisms to those they have demonstrated in rat
liver (Booth, Boyland and Manson, 1955) and that these conjugates are eventually
excreted bj^ the kidney. In the urine the glucosiduronic acids (but not sulphates
which are resistant to hydrolysis) are exposed to the action of hydroljdic enzymes
and thus free or/Ao-aminophenols are liberated. It is knovm that several of these
or</io-ammophenols, including 2-amino- 1-hydroxynaphthalene can induce bladder
cancer in mice and dogs (Hueper and Wolfe, 1937; Hueper, Wiley and Wolfe,
193S; Bonser, Bradshaw, Clayson and Jull, 1956; Allen «(., 1957; and Wallace,
1959).

In people not exposed to the chemical hazard it is suggested that orf/io-amino-
phenols derived from the metabolism of tryptophan may be one of the causative
factors in bladder cancer (Allen et al., 1957).

In a part of a series of investigations undertaken to examine this hypothesis
concerning the induction of bladder cancer, Boyland et al. (1955) demonstrated
that /^-glucuronidase activity is almost always increased in the urine of patients
with cancer of the bladder. They suggested that investigation of urinary f-
glucuronidase activity might be of value in the prognosis of such patients.

Because of the concentration of suitable cases available to the Bristol Bladder
Tumour Registry, it was decided to im’^estigate the significance of this urinarj
enzyme, employing a wide range of controls.

JIATEKIAE AND METHODS

Material

The control series fell into three main groups:

Group 1.— This group consisted of 32 healthy subjects aged 17-61 years witii
an average age of 33 years. Twenty-three (72 per cent) were male.

Group 2 . — There were 87 patients in this group with a wide range o gem
urinary'^ (g-u.) diseases other than tumours. The average age was 44 jmars vi
a range of 14-79 years.

URINARy /?-GLUCURONrDASE ACTmTY I'-"

Group 3 — Group 3 was a “miscellaneous” group of 120 patients n-ith an
average age of 51 years and a range of 16-82 years, with various diseases not
connected with the genito -urinary tract. Patients n-ith hone fractures were not
included but were assessed separately since they were found to have a significantly

raised enzjnne activity. , , -i. i

As the patients in Groups 2 and 3 were selected at random from the hospital,
the average age was lower than in the patients with cancer of the bladder. Two
sub-groups consisting of patients over 50 years of age were therefore isolated from
these main groups.

In 2a, the sub-group of the other genito-urinarj^ diseases, the average age was
59 years Wth 19 (91 per cent) male. In 3a, the sub-group of the “miscellaneous”
diseases, the average age was 60 years, of which 16 (47 per cent) were male.

Group 4. — Group 4 consisted of bladder carcinoma patients who came into
hospital mainly for review cytoscopy. There were 86 patients with an average
age of 63 years ranging from 38-81 years and of these 82 per cent were male. In
none of these was there any erddence of association nith the dye industrj^

Method

Twent3’--four hour specimens of urine were collected; the urine preservative
for about three-quarters of the investigation was 10 ml. benzene, but in the later
stages, m accordance with the revised method of Boyland et ul. (1957), this was
changed to 10 ml. of a 20 per cent solution of thymol in benzene. The thymol
with benzene had no effect on the enzyme level but had a greater bacteriocidal
effect than benzene alone. It was found that the enzjnne in urine of normal pH
was stable for at least seven days with the preservative at room temperature; this
was in agreement with the results of Boyland et al. (1955). Li the bladder cancer
group the urine was collected before cystoscopy. In some patients it was un-
avoidable that pre-operative drugs were given during the last hour or so of the
24-hour urine collection. In all groups it was noted whether the patient had been
receiving drugs or had been pyrexia! during the collection and whether there had
been any operative procedure within the preidous fortnight. The pH and specific
gravity of the urine were determined. In some cases the creatinine value was
estimated as a further check on whether the collection had been complete. Of the
101 specimens on which such determinations were made only eight had a creati-
nine value of less than 0-5 g./24 hours. This was taken as an mdication of the
level of accurac}’- in the urine collection. Unfortunately these estimations were
not carried out at the beginning of the whole investigation where collection errors
might be expected to have been greater.

Approximately 15 ml. of urine was centrifuged in a conical tube at 2000 r.p.m.
for at least 10 minutes. In a proportion of cases one drop of the deposit was exa-
mined under the microscope and patients with deposits containing more than 20
red blood corpuscles in the drop were considered to have excess red cells. Bed
cells have negligible ^-glucuronidase activity (Fishman, Springer and Brunetti
1948), but their presence in urine indicates the possibility of contamination with
seriun which has an activity of up to three times that of urine in normal subiects
The ^-glucuromdase activity of the supernatant fluid, and in some cases the
iinnary deposit also was determined by a slight modification of the method of

S’t.T?’ derived from the original method

of lalalay, Fishman and Huggins (1946). Duphcate samples of eiGier 1 ml of

108

P. J. W. LEWIS AND CONSTANCE H. J. PLAICE

supernatant unne or an appropriate volume of the deposit resuspended in distilled
water were incubated with 1 ml. acetate buffer {0-2 m, pH 4-5) and 1 ml phenolph
thalein mono-/5’-glucuronic acid (O-OS per cent in 10 per cent ethanol) in stoppered
tubes at 37° C. for IS hours. At the end of this time 1 ml. of urine was added to
the blank and a sufficient volume of 0-1 N NaOH was added to each tube just to
reach the pink coloration of the free phenolphthalein end-point. Then I ml. of
glycine buffer (0-4 m, pH 10-5) was added. The solutions were centrifuged for o
minutes at 2000 r.p.m. and the absorption at 550 m/i read in a Hilger spectro-
photometer. With urines of different buffering power it was necessary to add
different volumes of 0- 1 k NaOH to reach the end-point and allowance was made
for the different ffiial volumes obtained. The activity was expressed in the same
unit used by Boyland e( al. (1957), namety, 1 unit is the amount of enzyme liberat-
ing 1 jig. of phenolphthalein per hour at 37° C.

In 50 individuals the urine was investigated to determine the presence of heat
stable /^-glucuronidase inhibitors or activators. The enzyme activity of various
concentrations of urine diluted ivith distilled water was compared with the same
concentrations of urine diluted with boiled urine. The results were plotted
graphicall 3 >-, the percentage activitj? against the amount of dilution. In those
cases where the urine diluted with water gave a curve on the graph, while the same
urine diluted with boiled urine gave a straight line, it was assumed that an inhi-
bitor was present. Since these investigations were made on the unpurified enzjmie
the term inliibitor was used in its broadest sense. This inhibitor decreased in
concentration as the urine was diluted ndth distilled water but remained constant
when diluted with boiled mine (Boyland et al., 1955). The inhibitor index (P®)
was calculated as the ratio:

Unit of activity of 1 ml of a solution of urine diluted with

an equal volume of dis tilled water

Unit of activity of 1 ml. of a solution of urine diluted with

an equal volume of boiled urine

With regard to the reprodueibility of the method, the enzyme activity in 12
equal aliquots of urme urns estimated ; the mean value of the libera.ted phenol-
phthalein was 4-9 fig.jml. Tvith a standard deviation of 0-1 representing an error

of almost 3 per cent.

The accuracy of the method wms more difficult to determine. The cliief errors
arose from the different ionic and non-iom’c constituents and buffering powers o
the different urines. As Boyland et al. (1957) have showm, 0"2 M acetate u er
was sufficient to bring any urine rvitliin the normal range to a pH of 4-5-j4- . n
the investigation of urines wdth pH over 7-0 it was necessary to adjust the urine
to pH 4-5 with acetic acid before adding the pH 4-5 buffer. At the end ot le
experiment, after the addition of 0-1 N NaOH and 0-4 Ji glycine, it was
that the pH ranged from 10-1-10-5. Since the standard phenolphthalein cune
was determined at pH 10-5 there was, in some determinations, an error to
difference of the phenolphthalein colour intensitj^ at a pH differen rom
the standard. The magnitude of this error was determined in a series ot ;

The usual duplicate solutions were set up for incubation wuth an ,

taining 1 ml. urine, 1 ml. 0-2 iM acetate buffer and 0-4 M

At the end of the incubation time the same volumes of 0-1 x JSaun ana -
glycine were added to the extra tube as were to the duplicate tubes ai

URINARY ^-GLUCURONIDASE ACTIVITY

109

of a standard solution of phenolphthalein was then added to this tliird tube.
The colour developed in this tube was compared witli the colour developed by
the standard phenolphthalein in glycine buffer at pH 10-5. The percentage
difference was calculated. The mean percentage difference for the 61 urines was
6*9 with an S.D. of This difference included the error due to the different

final pH and the small salt and protein indicator error of phenolphthalein.

RESULTS

Before dealing with the range of /9-glucuronidase activity in the cases of cancer
of the bladder and the different groups of controls, the results of investigating
various factors which might be expected to influence this enzyme activity are
presented. A wide range of drugs was given to the patients as part of their treat-
ment. Excluding the steroid hormones, particularly cortisone, which appeared
to raise the enzyme activity, the general picture was that most drugs and anti-
biotics did not have any appreciable effect. As Boyland and Williams (1956) had
noted, patients with pyrexia often had a raised enzjTOe value.

Table I. — /^-glucuronidase Activity of the Urinary Sediment

(a) In normal subjects

/3-glucuronidoso activity

Initials

Age

Sediment

Sex

Units/ml.

original

urine

Supernatant

Units/day

G. R—

I\I.

P. K—

>»

003

K. C—

004

L. B—

004

JI. T—

007

J. K—

0-07

J. R—

»»

007

M. B—

0-09

102

C. P—

010

150

D. F—

010

166

JI. J—

Oil

176

F. K—

012

D. L—

0-12

M. B—

013

203

A. K—

0-14

199

V. B—

017

142

P. V—

018

435

J. A —

0-31

252

Units/ml.

urine

0-3G

0- 70

1- 37

0- 83

1 - 02
1-3
1-07
0-83
0-73

0- 69

1 - 0
1-02
1-15
1-05
0-51
0-94
0-87
0-9G

Units/day

7G5

830

1800

1910

1140

IGIO

1400

9G3

1300

1120

1G50

1150

697

1570

724

795

2100

784

(a) /i-glucuronidase activity in urinary sediment

Table I (a) demonstrates that the /9-glucuronidase activity of the small urinary
deposits found in 18 normal subjects ranged from 0-450 units per day and the de^
posit 111 women usually had a higher activity than in men. The enzyme level was

SmLTth? ?rs4^rn^t^4%^^^^^

behveen the activity hi the deposit and the activity of the”°supeZS

110

F. J. W. LEWIS AND CONSTANCE H. J. PLAICE

Patient

No.

1G5

405

404

377

196

166

243

154

164

Table I. ^‘Ql’uciiTQTtidciSG Activity of tliG UTliiavy Stdimt'iii
(b) In patients with cancer of the bladder

^-glucuronidase activitj^
*

Sediment

Supernatant

Age

Sex

units/mi.

original

urine

Units/day

Units/ml.

urine

-A ,

Units/day

0-48

1140

0-66

920

001

1-7

3900

. 65

0014

1-5

2860

0-05

1-74

1060

0-05

132

0-S8

2340

0-1

194

0-49

930

-f

0-23

426

0-85

1560

0-37

985

1-1

3100

IVith red corpuscles contamination

F. . I-OS 1439 1-77

2420

1-36

2860

1-02

2140

-)- = A tumour recurrence was found during the cystoscopy immediately following the urine
collection.

— = No reouirence.

(b) T//e presetice of ft-ghtctironidase inhibitors in the supernatant urine (Table II,
Fig. 1)

Of 52 men investigated from all groups, 45 (86 per cent) had an I®® of more
than T1 which was taken to indicate the presence of inliibitors. In 21 w'omen,

-pjQ, 1 . ^Inhibition of urinary ^.glucuronidase by substances present in urine.

O Diluted with distilled water.

X Diluted rvith boiled urine.

11 (52 per cent) had an I^® value of more than 1-1. If these results were due to
the presence of a single substance, this inhibitor was present m men as impiento
in cancer of the bladder (in 12 out of 13 cases) as in other *sease 28 out ol 54
or ill normal subjects (in each of the 5 subjects investigated). In ic iirJiie

Ill

URINARY /?-GLUCURONIDASE ACTIVITY

women the corresponding results were : cancer of the bladder (one out of two cases),
other disease (7 out of 12) and normal subjects (3 out oi t).

Table 11.— The Inhibitor Index of Supernatant Urine

Group

1. Normal subjects

Patient’s
No. or
initials

Sex

JSO

C. P— .

. 0-42

A. K—

. 100

M. C—

. 100

J. G—

. 110

J. H—

. 1-30

J. C—

. 1-40

M. B—

. 1-47

D. F—

. 1-50

G. R—

. 1-5C

J. B—

»»

. 1-70

P. V—

. 1-80

F. K—

. M.

. 1-90

2. Other genito-
urinary diseases

241

. F. .

0-03

371

. M. .

1-00

299

. F. .

1-00

139

. M. .

100

249

. F. .

1-06

251

. M. .

110

259

• tt

1-17

250

255

• it *

1-18

220

• ii *

1-20

237

* >> *

1-22

138

* a *

1-30

256

* ft •

1-30

—

•a *

1-30

257

* if *

1-36

231

* it •

1-41

121

• it •

1-43

422

1-44

High urinary pH

227

. F. .

1-20

198

. M. .

1-20

140

•ft •

1-30

112

• a •

1-44

• it •

1-54

Within 14 days

after operation

205

. F. .

0-80

240

• ft •

1-08

254

. M. .

118

135

• ft »

1-60

248

• tf *

2-25

Patient s

Group

3. “ Miscellaneous ”
diseases

4. Cancer of the
bladder

No.

Sox

Jto

F. .

0-76

238

M. .

1-10

193

423

223

1-3G

242

•

1-45

224

it •

1-GO

228

it •

1-00

195

2-00

219

2-43

F. .

2-G3

High urinary pH
13G . M. . 2-20

Within 14 da3's

after operation

245

. M.

1-05

247

• tf

1-17

204

• tt

1-66

199

• tt

1-73

202

' »»

2-00

203

' tt

2-0

196

. F.

0-79

165

. M.

1-00

154

• tt

1-17

• tt

1-18

405

• tt

1-27

166

• tt

1-43

152

* tt

1-48

404

• ft

1-50

279

' tt

1-55

377

• tt

1-57

• tt

1-70

378

• tt

1-78

With red cell

contamination

164

. F.

1-80

. M.

2-59

The urine from 3 women contained an “ activator ” of some kind, since on
dilution with distilled water the decrease in enzyme activity was greater than on
dilution with boiled urine (Fig. 2). In each of 4 normal subjects and 2 patients
on whom these investigations have been repeated, the inhibition or activation
originally demonstrated remained unchanged.

An attempt was made to correlate the enzyme activity with the phases in the
normal menstrual cycle. Four normal women between the ages of 20-30 years

112

F. J. W. LEWIS AND CONSTANCE H. J. PLAICE

Fig. 2. — Activation of urinary /3-glucuronidase b 3 ' substances present in some

women’s urine.

O Diluted with distilled water.

X Diluted with boiled lu-ine.

graph represents the total daily enzjnne output.

Menstruation.

. c ■ Contaminated by menstrual blood.

Time of ovulation.

113

URINARY ^-GLUCURONIDASE ACTIVITY

collected 24-hour specimens throughout one cycle and noted the time of ovulation
by recording waldng temperatures. Since the cycles Avere normal it was assumed
that peak oestrogen output occurred at about ovulation time and ]ust before
menstruation (Pedersen-Bjergaard and Tonnesen, 1948). The urinary ^-glu-
curonidase activity appeared to be unrelated to the phases of the menstrual cycle
except that in specimens grossly contaminated mth menstrual blood and tissue
the activity was elevated (Fig. 3).

Table III . — The fi-ghtcuro7iidase Activity in Infected Urine where the pH

is Within Norinal Limits

j9-glucuronidase

Patient

No.

activity

(units/ml.)

Organism

299

5-2

1-10

Tubercle bacillus.

174

5-2

1-77

Mixed conforms.

148

5-3

0-21

Conforms and B. proteus.

150

0-6

0-66

Conforms.

122

5-6

0-84

Conforms and staphylococci.

109

5-6

1-50

B. proteus.

112

5-7

0-96

E. eoU.

244

6-0

0-99

Mixed conforms.

130

6-0

1-29

E. coli.

110

6-0

1-51

Ps. pyoeyanea.

161

6-0

1-51

Fs. pyocyanea.

230

6-0

1-51

E. coli.

6-0

1-70

E. coli.

194

6-2

0-3

E. coli.

240

6-2

0-63

E. coli.

6-2

0-93

Mixed Avith E. coli.

256

6-2

1-05

Aerobacter aerogenes.

120

6-2

1-08

Ps. pyocyanea.

Aerobacter aerogenes.

242

6-2

1-11

iMixed coliforms.

218

6-3

1-10

Mixed coliforms and Staphylococcus aureus

274

6-4

0-51

E. coli.

6-4

0-66

Mixed.

253

6-4

0-90

E. coli.

226

6-4

1-41

Aerobacter aerogenes.

301

6-58

0-90

E. coli.

246

6-6

1-60

Conforms.

250

6-8

0-81

Conforms and Enterococci.

(d) The effect of alhaline conditions of the urine upon the p-glucuronidase activity
(Tables III and W)

With slight urinary infection where the pH was not raised above pH 7-0 the
enzyme activity was not significantly altered (Table III). In .5 patients Avith eancer
of the bladder the urine was grossly infected and in 4 cases the pH was raised to
a value of 9-0 or more. In these patients the urinary enzyme activity was in-
creased. In patients suffering from other diseases where the pH of the urine was
abnormally high, in several cases (6 out of 20), the enzyme aetmty was eleA^ated
(Table W)- The effect of incubating normal urine Avith B. proteus or E. coli before
the assay Avas variable, the enzyme aetmty being sometimes increased and some-
times decreased. "When the pH of normal urine was increased by the addition of
alkali the enzyme activity fell,

114

r. J. W. LEWIS AND CONSTANCE H. J. PLAICE

Table lY.—Vrinanj ft -glucuronidase Activity in Patients xoitli Urine of High pH

^-glucuronidase

activity

Disease

Patient

No.

Units/ml,

urine

— Sj

TJnits/day

Cancer of the

296

7-14

4-95

3,960

bladder

287

9-0

2-17

4,180

213

9-0

2-40

3,820

278

9-2

2-77

11,400

305

>9-0

3-28

3,180

Anj' other

222

7-2

0-93

1,311

disease

169

7-28

0-69

2,640

7-4

114

2,040

7-5

2-30

1,840

7-6

1-70

3,570

160

8-0

2-19

4,420

295

>8-0

108

>8-0

0-81

2,090

>8-0

1-18

2,840

227

>8-0

2-04

4,896

140

8-5

0-72

453

105

9-0

1-68

2,140

198

9-0

7-65

33,600 '

>9-0

0-72

630

248

>9-0

1-14

1,530

>9-0

1-57

2,155

>9-0

2-93

1,570

149

>9-0

3-96

10,300

>9-0

4-20

11,050

112

>9-0

9-45

5,760

Organism

Enterococci.

B. proteus.

Sterile.

Aerobacter aerogenes.
Non-haemol 3 ’tic strepto-
cocci.

Conforms -f Streptococcus
faecalis.

Mixed conforms.
Conform.

E. coll.

B. proteus.

Streptococcus faecalis and
B. proteus.
Diphtheroid.

Aerobacter aerogenes.

B. proteus.

(e) Urinary ft-gluctironidase activity after operation (Table V)

It was found that within 8 days after major operations performed on pcarts of
the alimentary canal the enzyme activity was significantly increased, which is
in agreement with the observation of BoyJand and Williams (1956). On the other
hand minor operations upon skin or fairly superficial muscle did not result in an
elevation of the enzyme activity. The effect of operations on the genito-urmary
tract upon the enzyme activity was variable.

Table V. — Urinary ft-glucuronidase Activity within 8 Days of Operation

Urinorj' p-glucuronidase activitj'

Type of
surgerj'
Minor .

Units/nd.

urine

“ t test against
normal subjects

Units/daj’

“ i ” test against
normal subjects

20 -

Mean S.D. S.E.

. 1-19 0-39 0-088

t >

t p

0 • 98 Between
0-4 and

Mean

1523

S.D. S.E.
569 127

'~t " p

0-52 0-6

Major .

13 .

, 3-72 1-68 0-466

0-3

8 • 08 Less than
0-01*

3534

1750 486

5-37 Less than
0-01*

S.E. = Standard error of the mean.
* Significantly different.

URINAKY /?-GLUCUBOKlDASE ACTIVITY

115

P -glucuronidase aclivitij in normal subjects, in cancer of the bladder and in. other
diseases (Table VI)

Group 1. — In normal subjects the mean activity /ml. urine was 1-05 units with
a standard error of the mean of 0-098 and the mean daily excretion was 1405
units/day with a standard error of 160. Boyland cl al. (1957) found the normal
range to be 0-05-1-2 units/ml. and when the daily outputs were calculated from
their published figures the mean Avas 1304 units/day with a range of 162-4890
units.

Table VI. — Urinary p -glucuronidase Activity in Normal Subjects and in Patients with

Different Diseases

“ l ” test ngninst “ t ” test ngainst

Average Units/ml. normal subjcct.s Unit.s/dny normal subjects

age

— ^ s

— —

— ^ \

Group (years)

Mean

S.E.

S.D.

Mean

S.E.

S.D.

. Xormal subjects

1-05

0-098

0-55

—

1405

160

893

—

. G.U. diseases (total age
range)

1-21

0-122

0-78

0-98

Between
0-4 and
0-3

2353

270

1707

2-8

0-01*

a. G.TJ. diseases (over 50
years of age)

1-31

0-227

1-04

1-14

Between
0-3 and
0-2

2251

441

2023

2-06

Between
0-05 and
0-02*.

. “ Miscellaneous ” dis-
eases (total age range)

1-10

0-084

0-02

0-82

Between
0-5 and
0-4

1719

141

1047

1-4

Between
0-2 and
0-1.

a. “ Miscellaneous ” dis-
eases (over 50 years
of age)

1-35

0-121

0-70

1-91

Between
0-1 and
0-05

1649

163

950

1-02

Between
0-3 and
0-2.

b. Fractures .

2-55

0-213

1-24

6-14

0-01*

2990

321

1697

4-55

Less then
0-001*.

Cancer of the bladder .

1-18

0-068

0-58

1-073

Between
0-3 and

1831

100

831

2-3

0-025*

0-2

S.E. = Standard error of the mean.
* Significantly different.

Table VIL— Daily Volume of Urine Excreted by Normal Subjects and
Patients with Different Diseases

2 .

2a.

3.
3a.
3b.

Group

Xormal subjects ....

Genito-urinarj- diseases (total age range)

Genito-urinary diseases (over 50 j-ears
of age)

“ Jliscellaneous ” diseases (total age
range)

“ Jliscellaneous ” diseases (over 50
years of age)

Fractures

Cancer of the bladder

Volume of
urine excreted
(ml.)

Mean

1419

2094

1824

S.E.

110

137

165

S.D.

577

901

755

1462

635

1360

625

1173

108

571

1739

601

“ t ” test against
normal subjects

Less than 0-001*.
Between 0 ■ 05 and
0 - 02 *.

Between 0-8 and
0-7.

Between 0-2 and

0-1.

Between 0- 02 and

3-52

2-108

0-356

0- 379

1- 529

2- 408

* Significantly different.

0 - 01 *.

116

F. J. W. LEWIS AND CONSTANCE H. J. PLAICE

Table VIII

Sd ir"" activity in subjects from the normal

and misceUaneous groups who had excreted over 1700 ml. urine rviththe

u^Sri^'feihses carcinoma of the bladder and rvith other geSito-

Group
1-f 3>1700

Mean

volume

urine

excreted

(ml.)

2150

Mean

daily

enzjnne

activity

(units)

2355

t tests

Urine volumes

24-hour enzj-me activity

Against
carcinoma
bladder group
.

Against

Against other carcinoma
g.u. diseases bladder group

t P

2-78 <0-01*

t P
0-253 0-80

2-37

0 - 02 »

Against othc
g.u. diseases

I —

t P
0-005 >0-1

(6) Comparison of the 24-hour 0023^0 activity in patients with carcinoma of the
bladder and other genito-urinar}^ diseases u'ho had excreted less than 1900 ml.
urine, mth the activitj^ in normal subjects.

Group

2-|-4<1900

“ t ” test against normal subjects

Mean volume
urine excreted
(ml.)

1323

Mean enzyme Urine volumes

activity , * ,

(units) t P

• 1687 . 0-890 Between 0-4

and 0-3

Significantly different.

24-hour

enzj-me activity

, * ^

t P

1-453 Between 0-2
and 0-1

Groups 2, 3 and 4. — The results in this section are from urine within the normal
pH range and not contaminated by excess red cells. During the urine collection
the patients were not suffering from pyrexia, had not undergone operations uatliin
the previous fortnight and were not having steroid hormone therap}’’.

In the original “ miscellaneous ” group it became obvious that patients with
fractures had an enzyme activity liigher than any other. These cases were there-
fore remoA'^ed and considered separately.

Apart from this (Group 3b) the /^-glucuronidase activity per ml. urine in any
of the groups (2, 2a, 3, 3a, or 4) urns not signiiicantl3’^ different from that in normal
subjects.

The dail}'^ enz3'^me output in both the total “ misceUaneous ” group (3) and its
sub-group of patients over 50 years of age was also not significantly different from
the normal Amine. The enz3'me output per day, howeA'^er, in the total “ other
genito-urinar}'^ diseases ’’{Group 2) and in its sub-group Avas significantly increased
as it was also in the group of cancer of the bladder cases (4).

It AAms suspected that the increased enzyme output per day in both groups 2
and 4 might be a mere statistical effect of the significantly larger volumes of urine
excreted (Table VII) as a result of the ^eater fluid intake encouraged in these tivo
groups of patients. Since it Avas very difficult to obtain reliable information about
the fluid intake the foIIoAving, admittedly arbitrar}’’, method Avas used to test such
a hypothesis. The daily enzyme output Amiues of indiAdduals in the normal and
“ miscellaneous ” groups who had excreted a Amlume of urine of 1700 ml. or more
Group (1 -f 3 > 1700), were statistically compared AAuth the enzyme actiAuty m
Groups 2 and 4 (Table VIH). No significant difference Avas found betAA'cen the

117

UBIKAHY /?-GLUCUBOKlDASE ACTIVITY

mean urine volumes in this (1 -f- 3 > 1700) group and the other genito-urinary
diseases (2), nor was there any significant difference in the daily enzyme excretion.
It was even found that the mean urine volume of group (1 + 3 > 1/00) was
significantly liigher than the mean volume of the carcinoma of the bladder group
(4), and, correspondingly, the daily enzyme activity in groups (1 + 3 > 1700)
was liigher than m the cancer of the bladder cases.

As an added check, all results from patients with carcinoma of the bladder and
other genito-urinary diseases who had excreted a daily volume of less than 1000
ml. (again an arbitrarj^ figure), were combined (group (2 4 < 1000)) and com-

pared with the enzyme activities in normal subjects (1). There was no significant
difference in the enzjmie output in these two groups : the mean volumes of urine
excreted were also not significant^ different.

The results from Groups 1, 2a, 3a and 4 indicated that, although the mean
values were higher in the older groups, age had no sigmficant effect on the urinaiy
/^-glucuronidase activity /ml. urine. In both the sub-groups of patients over 50
years of age the mean volumes of urine excreted were less, but not significantly,
than in the corresponding main group, and the mean daily enzyme activities were
also lower.

In the fracture group, 3b, both the activity per ml. of urine and the daily
enzyme output were significantly raised compared rvith the normal values for at
least 10 da3"s after the fracture (Lends and Plaice, 1959). As the mean volume of
urine excreted was lower than normal it was assumed that this was in fact a true
difference.

DISCUSSIO^■

The enzyme concentration per ml. of urine is an indication, at anj’’ particular
time, of the possible hydrolytic opportunities, but the length of time the bladder
mucosa is exposed to the carcinogens is another factor wliich must play' a part.

The enzyme activity' per ml. of urine was not significantly increased in the
cancer of the bladder patients, which is not in accord with the results of Boy'land
et al. (1955). When, however, the enzyme activity' per day' was considered, both
these and the other urinary tract diseases had significantly' increased activity'
compared with the normal value. This increase was not only' not specific for the
cancer of the bladder patients but it appeared to be a statistical consequence of
the greater volumes excreted. This apparent relationship between the volume
excreted and the daily enzyme output is difficult to understand.

Boy'land and his colleagues (1955) found no //-glucuronidase inliibitors in the
urine whereas in the present series 86 per cent of the samples from men and 52
per cent from women had an inhibitor present. The presence of an endogenous
/?-glucuronidase inhibitor in many types of tissue and in serum is well known
(WalkCT and Lev\'y', 1953) and an enzyme inhibitor in urine has been demonstrated
(Abul-Fadl, 1957). Lem'y' (1956) points out that an important cause of inhibition
ot hyffiolysis of a particular glucuronide is the presence in urine of other glu-
curonide conjugates. This will in any case introduce errors of unknown magnitude
m the determination of urinary ^glucuronidase activity. It is of interest to note
that under the experimental conditions used in tliis study, the urine of some
women contained no inliibitors and appeared to have “ activators ” If d-glu
curonidase plays an important role in releasing bladder carcinogens it is therefore

118

S'. J. W. LEWIS AND CONSTANCE H. J. PLAICE

difficult to understand why women have a lower incidence of bladder cancer tlnn
men unless there is some difference between the sexes in the length of time that
the carcinogen can act upon the bladder epithelium.

The method used in this work and in the investigation of Boyland et aj. {19551
entads a dilution of urine with acetate buffer and substrate. This dilution of any
mJiibitor or ‘ activator ” does not give an accurate picture of the enzyme activity
in the bladder. alker and Levvj (1953) discussing an endogenous inhibitor
affecting rat-liver /?-glueuronidase actmty in in vitro investigations state that it
IS by no means certain that the inhibitor has any effect in vivo. To studj’' tlie
enzyme in the liver they homogenised the tissue, thus disrupting the cell con-
stituents and perhaps causing contact of enzyme and inhibitor which would
not naturally occur. In urine however, it is more likely that the inliibitor is as
active in the bladder as it is in vitro. The enzyme activity in the bladder is usually
less than that estimated in vitro at an optimum pH of 4-5. It is possible that the
inliibitor or activator action differs at the pH of the urine in the bladder from
that under the experimental conditions (Smith and Jlills, 1 953). Odell and Fishman
(1950), investigating the ^-glucuronidase activit 3 '^ in human endometrium, found
that there was a variation of activity during the menstrual cycle. They found
that the lowest values of the enzyme are immediatelj^ preceding and following
the period of the menses and the highest values occur in the interval between. It
seemed possible that these changes in endometrial activitjf might be reflected in
the urinary output although Fishman et al. (1951) found no consistent correlation
between the blood enzyme activity and the phases of the cjmle. The function of
/?-glucuronidase is still obscure. Not onlj" is it possible that the enzyme might
be involved in the synthesis of oestrogen and pregnanediol glucuronides, although
this is now considered unlikely, but it might also be involved in structural pro-
liferation of the uterus in the phase before ovulation and in the production of
mucin in the secretory phase. In women, cancer of the bladder usually occurs in
the post-menopausal stage and the enzyme determinations would not be affected
by any cyclical changes. It wms thought at the beginning of this investigation that
in the control groups, where there was a greater number of younger Avomen, the
menstrual cycle might have affected the enzyme actmty. If, however, /?-gIu-
curonidase plays a role in any of the metabolic processes during the menstrual
cycle it was not evident in the urinary activity of the small series inAmstigated.

With regard to the increased urinary enzyme actiAuty found after some opera-
tions and apparently associated Avith fairly large amounts of tissue proliferation,
Levvy’s revicAv (1956) must be borne in mind in Avhich he emphasises that there
are many results obtained bj^ different Avorkers which do not support the general-
isation that /9-glucuronidase actmty is invariably related to tissue grou4h.
Although this might be the reason for the elevation during the healing of fractures
there may be a relationship between hydrolj^sis of chondroitin and other mucopoly-
saccharides and the enzyme actmty, as suggested by LevA^y (1956).

It is possible that the discrepancy between the results of Bojdand et at. (I 0 1
and those reported here is due to a difference in the stage of the disease wien le
patient was investigated. The patients investigated at Southmead Hospital
(except for three of the cases in Table IV) were fairly fit and Avere able to resume
normal activities upon discharge after review cystoscopy. In advanced, inopera
cases, even where the pH of the urine is normal, it is feasible that contamination
of the urine from the tumour tissue which, Boyland and his colleagues ( ■ )

URINARY /^-CtUUCURONIDASE ACTIVITY

119

have shovTi to contain high enzyme activity, would cause an increased urinary
activity. Our results on the enzyme activity of the urinary sedimeirts from
patients with cancer present in the bladder were inconclusive. On the other hand,
Boyland’s cases may also have had significant bony metastases, another differential
feature from ours. It was considered possible that as new bone formation was
associated with an increase in the urinary' glucuronidase activity, some forms of
metastases in bone might have a similar effect. To date a small series of results
in patients with such metastases (unpublished) has not been helpful, possibly on
account of the steroid hormone therap 5 ’^ given.

Since this was written our attention has been drawn to two recent papers
concerned with urinary /?-glucuronidase activity in patients with cancer of the
bladder. Ohkubo, Sonoda and Kusonoki (lOfiS) in an investigation of the enzyme
values of serum, urine and tissue of patients with urologies! diseases found that
in 6 out of 7 cases of bladder cancer the urinary enzyme values were higher than
in 16 normal subjects. Mattea and Pietra (1959) observed that in 30 subjects with
industrial cancer of the bladder 29 had increased enzyme activity in urine diluted
ten times compared with the activity of similarlj'^ diluted urine of 4 normal subjeets.

The absolute values obtained by the authors of these two jiapers cannot be
directly compared with each other, with the results of Boyland et al. (1957) or
■\vith the results in the present paper, since experimental factors such as urine and
substrate concentration differed.

SUMMARY

1. Urinarj’’ /?-glucuronidase activity was estimated in patients with cancer of
the bladder and in a large number of controls, including patients with other diseases,
in addition to normal subjects.

2. ^-glucuronidase inhibitors were found in 86 per cent of the men and 52 per
cent of the women investigated for this effect.

3. The enzyme activity per ml. urine as well as the daily output were both
significantly higher than normal in some cases after major surgery and also, for
at least 10 days after fractures.

4. Apart from this the enzyme activity per ml. urine was not significantly
different in cancer of the bladder from that in normal subjects or in any of the
diseases investigated.

5. The 24-hour output of the enzyme was significantly increased in cancer of
the bladder and in other genito-urinary diseases. Data are presented which
appear to correlate this increase with the increased volume of urine excreted by
such patients.

'""ork was supported by a grant from the British Empire Cancer Campaign
H. J . P.). We wish to thank Mr. Ashton Miller who inspired this work,
i r. Mitchell and Mr. Slade for their kindness and help in putting this material
a our disposal. We also would like to thank Professor Gordon Lennon, University
epartment of Obstetrics and Gynaecology, Southmead Hospital, for provision
Lptio greatly indebted to Dr. G. Herdan, Statistician,

heln Preventive Medicine, the University of Bristol, for his valuable

, statistical analysis. We also wish to thank the nursing and ancillary
01 the hospitals concerned for their co-operation.

120

S’. J. W. LEWIS AND CONSTANCE H. J. PLAICE

REFERENCES

Abul-Fadl, M. a. M— (1957) J. din. Path., 10, 387.

Allen, M. J., Boyland, E., Dokes, C. E., Horning, E. S. and Watson, J. G.— (1957)
Brit. J. Cancer, 11, 212.

Bonser, G. M., Bradshaw, L., Clayson, D. B. and Jull, J. W.— (1956) Ibid., 10, 539.
Booth, J., Boyland, E. and IIanson, D.— (1955) Biochem. J., GO, 62.

Boyland, E., Gasson, J. E. and Williams, D. C.— (1957) Brit. J. Cancer, 11, 120.
Idem, Wallace, D. M. and Williams, D. C. — (1955) Ibid., 9, 62.

Idem AND Williams, D. C. — (1956) Rep. Brit. Emp. Cancer Campgn., 34, 40.

Fishman, W. H., Kasdon, S. C., Bonner, C. D., Fishman, L. W. and Hombdbgeb,
F. — (1951) J. din. Endocrin., 11, 1425.

Idem, Springer, B. and Brhnetti, R. — (1948) J. biol. Chem., 173, 449.

Hueper, W. C., Wiley, F. H. and Wolfe, H. D. — (1938) J. industr. Hyg., 20, 46.
Idem AND Wolfe, H. D. — (1937) Amer. J. Path., 13, 656.

Levi'Y', G. a. — ( 1956) ‘ Vitamins and Hormones ’, 14, 289 (edited Harris, R. S., Marrian,
G. F. and Thimann, K. V.). New York (Academic Press Inc.).

Leivis, F. j. W. and Plaice, C. H. J.— (1959) Nature, 184, 1249.

SIattea, E. and Pietra, E. — (1959) Tumori, 45, 86.

Odell, L. D. and Fishman, W. H. — (1950) Amer. J. Obstet. Gynec., 59, 200.

Ohkubo, T., Sonoda, T. and Kusunoki, T. — (1958) Urol, int., 7, 167.
Pedersen-Bjergaard, K. and Tcnnesen, M. — (1948) Acta endocrin., 1, 38.

S»nTH, E. E. B. AND IMills, G. T. — (1953) Biochem. J., 54, 164.

Talalay, P., Fishjian, W. H. and Huggins, C.— (1946) J. biol. Chem., 166, 757.
Walker, P. G. and Leiwy, G. A. — (1953) Biochem. J., 54, 56.

Wallace, D. M.— (1959) ‘ Neoplastic Diseases at Various Sites ’, 2, ‘ Tumours of the
Bladder ’. Edinburgh and London (E. S. Livingstone, Ltd.).

121

ihe antigenic structure or a transplanted sarcoma

r. O’GORMAN AKI) Z. B. MIKGBSKA
From the Dcjmtmcnl of Palhohuj,,. Guifs Hospital .S'.AM

Kcooivocl for puUliciMion Dcrouibor 31. Hir.n

, , oUr.«-n In TiosscRS activity when

iluRKE iso-antisera have been si o - 1 presence of active corn-

brought into contact wWi the targe ^cc ’ ]Qf,o). These authors have

plement (Gorer and O’Gorman. IhoG; ^ Borman,

shown that the cytotoxic iso-antibo les ar . controlling the fate of

antigens, which is known to be the gerie.s of

homografts in mice. The H-- has been summarised by Gorer and

alleles, and our present knowledge of it has been

Mikulska (1959). i ho stroneW cytotoxic for normal and

The iso-antibodies have boon sbonu to bo st^ /target cells being killed

malignant mouse leucocytes, up to 100 ] been found by these authors

(Golt and O'Gorman, lhr,6). Other ‘ho 0,H

to be less sensitiye to the cytotoxic actn ity ^ target

sarcoma BPS is only partially band, ha^ <o.ni to ta

cells djdng after contact. Sarcoma I, o , ;t.r,.nntisera
completely insensitive to the cytotoxic activi y , antibody than in the

no more Ln-rdable cells in the suspensions treated '^^^^^thm^suscep
normal serum controls (about 5p^r cm^ m 1^^^^^.^) cytotoxic anti-

of three ascites tumours bal, Bt » anu tue nnti-BP8 which contains an

serum is shonm in Table I. The serum was ( i, common to all three

antibody against an H-2 component, antigen E. which is commo

tumours (O’Gorraan, 1960).

Table l.-Cytotoxic Effect upon Three Tumours

Target

cells

EL4

BPS

Sal

Percentage non-viable cells at anti.serum*
dilutions ol :

Controls , 8 j

* Antiserum was BALB/c anti-BP8,

It seemed possible that this PheTarcoma

various tumours and normal liver, and theri i ra e ^ H i,„c formed

baemagglutinating activity, was undertaken to test this ™

part of a thesis submitted to the University of London for the degree of Doctor of
I'ledicine.

122

F. o’gORMAN A3ST) Z, B. MIKULSICA.

MATERIALS AND METHODS

Serological techniques. ~l. Tlie Jiuman serum ; dextran sj^stem described bv
Gorer ^nd (1954) B^as used for the experiments using haemagglutination.

rr^ 1 technique described by Gorer and O’Gorman (1956) and

modified by 0 Gorman (1960) ivas used for the cytotoxic experiments. Giiinea-
pig serum was used to provide complement and trjqian blue (1/2000 in Rinser’s
solution) ivas the dye. ®

ifice^Animals were used from the four inbred lines Strain A, BALB/c C,H
and C57B1 and also Gie so-called E-B-f (H-2g) cross-over, all maintained in
the laboratory of Dr. P. A. Gorer.

Timours.~Two sarcomata were used, Sarcoma I (A strain) and BPS (C,H)
and one leukosis, EL4 (C57B1). All three are ascites tumours and -n-ill sometimes
groiv progressively if inoculated intra-peritoneally in large doses, in foreign strains,
although they are strain specific when smaller doses are given subcutaneouslj'.
Suspensions of these tumour cells were made in Ringer’s solution in the case of
the leukosis and in 3 per cent sodium citrate for the haemorrhagic sarcomata, by
puncturing the distended abdomen B'ith a sterile Pasteur pipette, aspirating the
contents and ejecting the fluid into the diluent.

RESULTS

A. Experiments using cytotoxic technique

Sera of the t 3 q)e BALB/c anti-CjH contain the c 3 dotoxic antibodies anti-E,
anti-K and anti-D'^. Such a serum, BALE /c anti-BP8, was diluted 1 ; 4 and
absorbed for 75 minutes at 37° C. with equal volumes of BALB/c liver, and packed
cells of EL4, BPS and Sal ascites. The absorbed sera were tested on EL4. The
only antibod 3 ’’ udiich was being tested was therefore anti-E. The titre of the
unabsorbed serum was 1 : 64, and that of the serum absorbed witli BALB/c liver
was also 1 ; 64. No residual cytotoxicity was found after absorption ivith EL4
and BPS, but absorption with Sal left C 3 dotoxic activity up to a titre of I : 32.
Both EL4 and BPS had therefore absorbed anti-E, as would be expected. Sal,
on the other hand, had not absorbed anti-E and the tumour cells were presumed
to lack E.

This experiment gave no uiformation as to the amount of anti-E which would
be absorbed b 3 '^ normal A cells, and therefore a further experiment v’as performed
in which the same BALB/c anti-CgH serum was absorbed ivith C liver, A liver and
Sal and tested on EL4. The antibody is again anti-E. On this occasion the
titre of both the unabsorbed serum and that absorbed ivith C liver ivere 1: 128.
No cytotoxicity was left after absorption with A liver, but absorptmu uath Sal
left a cytotoxic titre of 1 : 32. It was concluded that Sal contains less E antigen
than does A liver. However, absorption with Sal lowered the titre by 2 tubes
compared ivith BALB/o liver. This might indicate that Sal is not totall 3 ’; lacking
in E ; on the other hand, this absorption may be due to the presence of norma
A strain cells (probably mainly red blood cells) in the Sal ascites.

B. Experiments using haemagglutination technique

A sample of BALB/c anti-EL4 (containing anti-D'’ and
at various dilutions with one equal volume of A hver. At a dilution of 1 . 3-

a^:tigenic strl'ctuke or a kakcgma

12 a

the nnti-E .vas abeorhed a„d no hnemagBh.linalion wna deleclod for A alnd., or
G H red blood cells. At. this dilution absorption wilb Sn left n (i u. of 1 . - 1.
for C,H cells and with BALB/c liver left a titre of 1 : 40110, boll, doe 1.. I

MLl. enntain-

anti'E.

A serum was produced in the E-Ed- (H-2g) crossmmr npinst EL-1, enntam-
anti-E and anti-K'’. When tested against A and C.^E red cells, onl\ nnti-L i.
Tiao niinnonrRpfl Rnriiui uaYC a titre of 1 : lO.aS-l for A cells and 1 ; ID.Xi

mg
involved.

A ceils and less than 1 ; 16 for CgH. i o ,

In an attempt to get some estimate of the amount of E jirc.sent on the hnl
cells the absorptive power of Sal for anti-E was compared with that of dcci casing
volumes of A liver. To do tins satisfactorily a minimum amount of antibody must
be used, so that one is working at the end of the titre. When I'j — B-|- anf i-li/L-l
was diluted 1 ; 256 and absorbed with 0-1 volume of A liver the anti-E remaining
only agglutinated A strain eiythroej-tes in the first tube. i.e. at. a dilution of
1 ; 512. 1-0 volume of Sal, however, absorbed more anti-E than 0-1 volume of A

liver, for there was no residual agglutination of A cells at 1 ; 512.

Tins same serum was then diluted 1 : 64 and absorbed with O-l or 0-2 volume
of A liver and with 1-0 volume of Sal. In tliis case the titre for A cell.s was
reduced from 1 ; 4096 for the unabsorbed serum to 1 ; 128 by absorption with 0-2
volume of A liver and to 1 : 256 with 0-1 volume. LO volume of Sal lowered the
titre to 1 : 128. It was concluded that Sal packed ascites cells contain only
about 20 per cent of the E antigen which is contained in normal A strain liver cells.

Similar experiments were done to test for the presence of the K and D antigen.s
on Sal cells. Sera produced in the (BALB/c x C57Bl)Fj against A strain tumour
contain only anti-K. Such a serum was absorbed with one volume of A liver or
Sal at various dilutions. When diluted 1:4, 1 : 8 or 1:16 absorption with A
liver reduced the titre from 1 -. 1024 for unabsorbed serum to 1:16 for tlie first
dilution and less than tliis for the other two. Sal reduced the titre to 1 : 512,
1 ; 256 and 1 : 128 for the dilutions 1 : 4, 1 : 8 and 1:16 re.spectively. This show.s
that Sal contains much less K antigen than does A liver.

The results of testing for antigen D showed no significant difference between
the amount on Sal and A liver cells. The serum used was made in the (G 3 H. X
C57Bl)Fi against A strain tumour, and contained only anti-D. The antibody was
absorbed and tested in the same way as anti-K. Both Sal and A liver left similar
titres at all three dilutions. They were 1 : 64, 1 : 16 and 1 : 32 after absorption
at 1 ; 4, 1 ; 8 and 1 ; 16 respectively.

The results of these a,bsorptions are expressed graphically in Fig. 1 . They
show that Sal packed ascites cells contain about 20 per cent of the E antigen and
mudi less K than normal A strain liver cells, but approximately the same Imount

UlSOUSSIOX

‘I'' ‘ypes of reaction to tumour

theiJ transplantable tumours based on

The flSSS. 1 St tli'-itliog them into tlnee classes

flrst moludes tumours which arc completely susceptible to the action of anti

124

p. o’goeman and z. b. mikulska

body , such as EL4. The second comprises tumours which are partially susceptible
such as 6C3HED, which tend to respond to the action of antibody by enhanced
rather than inliibited growth. Sal falls into tliis group. The tliird group are
those which are quite insensitive to the action of antibody, for example the aLno-
carcinoma D1905.

ABSOHPTION

TITRE

ANTIBODY

HESroUAL ANTIBODY TITRE FOR A STRAIN R. B. C. s

ABSORBED J_ _L _L _L 1 I 1 I

WITH 16 32 64 128 256 512 1024 2048 4096 8192 IMM

BALB/c LIVER
A LIVER

EQUAL VOLUMES OF LIVER OR S A 1 PACKED CELLS WERE USED FOR ABSORPTION
UNLESS OTHERWISE STATED
Pjq. ]. — ^Absorption of iso-antibodies bj' Sal.

The results of the extensive absorption experiments reported above show that
Sarcoma I growing in the ascites form in the Strain A mice inaintained in Un
Gorer’s laboratory possesses about 20 per cent of the antigen ®
corresponding amount of normal A liver ; it also possesses less of ^
than does A strain liver, but about the same amount of anfagen D There appe
to be three possible explanations for this antigenic loss. The firsV b^s eond
Sal cells are deficient of the antigens by the same amount, f

that some 80 per cent of the cells are wholly E and

ing 20 per cent carry the normal amount; similarly K might be parfciallj missing

AXTIGENIC STHUCTURE OF A SARCOMA

12 .')

from all the cells, or totally missing from some. The iJiircl ])ossiI)iIi(,y is that the
sarcoma cells arc all deficient of the.se antigens and their apparent prc.sencc i.s due
to contamination of the ascite.s willi normal erythrocytes and leucocytes having
normal antigenic con.stitntions. We have no definife evidence for or against
any one of these theories hut as Sal ascites alwaj-.s contains a ])roportion of host
erythrocytes and leucocytes it seems reasonable to deduce that Sal cells are wholly
deficient of antigens E and K. and (hat the small amounts found are due to the
normal cells in the ascites. We can only speculate upon the po.ssiblc rclation.shij)
between this antigenic lo.ss and (he rcndine.ss with whicli the growtli of Sal may
be enhanced by the action of antibody on the one hand, and on other, the insensiti-
vity of Sal cells to the /?/ vitro to.vic effect.

Kaliss (10.5S) believed that immunological enhancement is due to a direct
effect of antibody upon the tumour cells. 'I’his is evidently' not a toxic but. rather
a stimulating effect, so that the tumour is altered in some way and is thus better
able to withstand and overcome the host’s defensive reaction. Sarcoma I may
be insensitive to cytotoxic antibody because of its antigenic deficiencies, but this
need not preclude a stimulant effect of the antibody on the cell and stimulation
of an antigen deficient cell by antibody' Jinay' lie at the root of the jnechanism of
immunological enhancement.

SUMMARY

Experiments have been reported which have shown, by means of graduated
absorption of iso-antisera, that suspensions of Sal show marked deficiencies of
the H-2 antigens E and K, but no deficiency' of antigen D. It is possible that
these deficiencies are connected with the lack of sensitivity' of Sal to cytotoxic
antibody and the ease with which the growth of the tumour may' be enhanced.

The authors wish to express their gratitude to Dr. P. A. Gorer for his help and
advice during this investigation.

REFERENCES

Goker, P. a. and JIikulska, Z. B.— (1954) Cancer Res., 14, 651.— (1959) Proe. Ron.
Soc. B, 151, 57.

Idm AND O’Gokman, P.— (1956) Trans-pl. Bull., 3, 192,

Rttiss, N.-_( 1958 ) Cancer Res., 18, 992.

Goeman, P. — (I960) Bril. J. Cancer, 14 (in pre.ss).

SELL, G. D. — (1957) Cancer Research, 17, 2.

126

EFFECT OF LETHALLY DAMAGrED TUMOUR CFTT*^ ttpaat 01^
OEOWTH OP ADMIXED VIABLE (®LLS IN dSpSn ChSib™

K. J. SEELIG AND L. REvESZ

From the Institute for Tumour Biology, Karolinska Institutet Medical School,

Stockholm, Sweden

Received for publication December 4, 1959

It has been shown that the proliferation of a small viable tumour graft is
stimulated hj’- the presence of irreversibly X-ray damaged tumour cells (Revesz,
1958) or viable but genetically incompatible cells (Klein and Klein, 1956).
Histological examination (Ringertz, Klein and Revesz, 1959) showed an enhanced
granulation tissue formation in and aromid the implant. The intensity of this
reaction was parallel to the stimulating effect of X-ray damaged, geneticalty
incompatible, and heat-killed cells, respectively. This would indicate that the
stimulating function may depend on the formation of a proper tumour bed. In
addition, a direct “ feeder ” effect (Puck, Marcus and Cieciura, 1956) may also
play a certain role since heavily irradiated cells were stimulatory even in the case
of freely suspended ascites tumour cells (Revesz, 1955; Scott, 1957; Mazurek
and Duplan, 1959).

The diffusion chamber technique of Algire, Weaver and Prehn (1954) permits
the isolation of the graft from direct contact with host cells. Filter membranes
with adequately small pores permit the diffusion of soluble materials but prevent
the outbound passage of graft cells and inbound movement of the host cells.
Grafts of various kinds have been shorni to survive and proliferate for long periods
of time under such conditions (Algire et al., 1954 ; Shelton and Rice, 1958 ;
Amos and Wakefield, 1958 ; arid others).

In the present paper the growth of untreated ascites tumour ceils was measured
alone or in the presence of lethally damaged cells vdthin diffusion chambers in
an attempt to clarify the role of the cellular host reaction in the stimulation
phenomenon.

MATERIALS AND METHODS

Mice : Various F^ hybrids were used, obtained by mating males of the A.SW
strain (SneU, 1955) with females of the strains G3H/K1, DBA/2/IU and C57BL/K].
The animals weighed 20-26 g. and were 2—4 months old. They were maintained
on a standard peUet diet which, together with drinking water, was available

ad libitum. ,

Tumours: The Ehrlich/Sto ascites tumour (Klein and Revesz, 1953) pro-
pagated in hybrid mice was used in all experiments except one with the LRIO
ascites lymphoma of the DBA/2 strain (Law et al, 1949). In some experments,
the following two tumours were admixed with cell suspensions of the Elwhcli
carcinoma : Ascites sarcoma MClM of strain G3H origin (IGlein 1051) and the
solid fibrosarcoma MSG induced by methylcholanthrene m an A/Sn mouse.

The ascites tumours were propagated by weekly inteperitoneal trans
0-1 ml. ascites fiuid diluted 1 : 10 in Ringer’s solution. The sohd neoplasms

EFFECT OF DAiNIAGED TUMOUR CELLS ON VIABLE CELLS

127

suspended in Ringer’s solution after having been forced through a stainless steel
screen and were propagated by subcutaneous transfer.

Irradiation : X-rays were generated in a Siemens X-ray machine at 185 kv
and 15 mA, and were filtered by 1 mm. Al. Irradiation of the tumour suspensions
was performed in vitro m a sterile flat-bottomed plastic irradiation chamber "with
an inside diameter of 26 mm. and a height of 24 mm. The suspensions, each
approximately 5 ml., were irradiated with 12000 r at a rate of 425 r/min ; the
distance from the focus of the X-ray tube to the bottom of the vial was 29-5 cm.

Chambers : The diffusion chambers used in the present investigation were
designed after the chambers of Algire et al. (1954). They consisted of two parts :
(1) a “ cup ”, constructed of a disc of HA millipore filter (Millipore Filter Corp.,
Watertown, Mass.) sealed to the bottom of an acrylic cylinder (material manu-
factured by R. Daleman, Ltd., London) having an outside diameter of 16-8 mm.,
an inside diameter of 14-5 mm. and a height of 5-0 mm. ; (2) a “ lid ”, ivith a
construction similar to that of the cup, but with larger dimensions, namely 19-5
mm. outside diameter, 17-0 mm. inside diameter and 5-0 mm. height. When
adjoined they composed a closed chamber having a volume of 0-8 ml. In one
experiment (with L1210 lymphoma) chambers of half of this volume were used ;
they had the same diameter but a height of only 2-5 mm.

The HA millipore filter was employed in all experiments with an average pore
size of 0-45 ji. The filter was sealed to the acrylic cylinders with acetone in which
filter material had been dissolved (about 4 filters to 10 ml. acetone). The cups
and lids were sterilized by immersion into 70 per cent alcohol for 6 hours. Sub-
sequently they were placed in sterile Petri dishes and dried in a desiccator.

Transfer procedure

Transfer of the tumour cell suspensions into the chambers was performed
under aseptic conditions in a room assigned for sterile work. The cups, usually
10 of them at a time, were placed in a row on a sterile metal plate and 0-2 ml.
ice-cooled tumour suspension, appropriately diluted in Ringer’s solution, was
pipetted into each cup. The lids were then drawn over the cups with a forceps,
the chambers were inverted and their two parts were sealed by a commercial
fast drying lime (Karlssons Klister, AB Klarre, Stockholm) at their junction.
Usually thirty to forty chambers were prepared in an experimental series by a
single operator. The closed chambers were kept in ice-cooled Ringer’s solution
until insertion into the peritoneal cavity of the mice.

The animals were anaesthetized with 0-006 ml. per g. body weight of a 1 per
cent Nembutal solution injected intraperitoneally. The abdominal skin was
washed with a cotton wool pledget moistened with alcohol, and an incision of about
L cm. length was made. The chamber was inserted into the peritoneal cavity and
the abdomen was closed by a continuous silk peritoneal suture and agraff skin
clips. Depending upon the nature of the experiment between 2 and 6 hours

k'St fbSit- a

Sampling procedure

Each animal was Icillcd bv cervical dislooatinn n

the chamber .as carefully ’iensoved’

128

K. J . SEELIG AND L. RBVESZ

accumulated host cells was rubbed oflF the external surfaces by a filter pauer
Subsequently, the chamber was either used for the quantitative detenninatioj of
1 s cellular content or for sterility and inability tests, respectively. Different
c lambers inoculated with the same pool of cells were assigned for these two pro
cedures m a random way. ^

Quantitative sampling : Each chamber assigned for quantitkive determination
oi its total cell content was covered ivith a commercial nailpolisli over its entire
surface in order to trap the host cells adhering to the outer surface and to prevent
them from intermixing with the chamber contents. The chamber was fixed in
a small holder apparatus and opened on one side by cutting it with a pointed
forceps around the edges of the millipore filter. After the incubation periods
used in this study, all chambers were found to be completely filled with a viscous
fluid in which clots were frequently observed.

Following opening the entire chamber was submerged into a flat-bottomed glass
vial containing 4 ml. of aqueous solution of 0-1 ax citrate and 0-5 per cent crj'stal
violet. The vial was plugged udth a rubber stopper and shaken at a frequency of
about 1200 oscillations per minute and an amplitude of 0-8 cm. for 5 minutes in
a microid flask shaker (Griffin and Co, Ltd., London). This treatment dissolved
occasional clots and provided a homogeneous suspension of stained cells. Micro-
scopical examination of the filters indicated that the shaking treatment was also
effective in removing all cells from the inside of the wall of the chambers.

The cell concentration of the stained suspension was determined in undiluted
or diluted (1 : 10) aliquots in a Buerker haemocjijometer. At least 50 cells were
counted in 4 x 1 ram® undiluted fluid. In difihsion chambers ufith a volume of

0-8 ml., this cell number corresponds to a total population of 6 X lO-* cells. The
population size of chambers containing less than this number was not determined.

The proportion of cells containing nuclei stainable udth crystal violet was
estimated by differential counts on the material in the haemocjdometer. At
least 200 cells were counted. Absent or indistinct nuclear staining with dispersed
and/or fragmented chromatin debris was taken as evidence for irreparable cellular
damage. The proportion of such cells was taken as the minimum limit of non-
viable cells in the population.

Sterility and viability tests ; The chamber was removed from the peritoneal
cavity and cleaned from the coating capsule. Fixed in the holder apparatus, one
of the chamber membranes was perforated by the point of a Pasteur-pipette.
A small drop of the chamber contents was transferred into a bouillon broth and
incubated for three days at 37° C. If bacterial growth was obtained, all chambers
inoculated from the same pool were rejected. Usuallj^ an experimental series
consisted of six chambers out of wdiich two were used to test bacterial

contamination. i . t

In addition to the sterility test, the same chamber was also used to estimate
the approximate proportion of viable cells by the procedure of Schrek (193 ).
In order to bring all tumour cells into a homogeneous suspension the open chamber
was transferred into a glass vial, containing 4 ml. ice-cooled Locke s solution
wLich ivas shaken for 5 minutes. The possible damage caused by tins treatment
to viable cells was not determined. The eosin-unstamed fraction ot cells ii
therefore considered as a minimum estimate of the originally viable fraction.

A comparison of the results obtained by the crystal violet procedure, iisin
nucLr staining as criterion of viability, and the Schrek test, shows a positive

129

effect of damaged TUJIOUE cells on A'-DVBLE cells

correlation although the Schrek test gave usually a lower estimation of the size of
the viable fraction (Fig. 1).

Percentage of cells with poor nuclear stainability
by crystal violet.

Fjq 1 Xhe percentage of Sohrek-positive cells as correlated with the proportion of cells wit h

poor nuclear stainability in the crystal-violet test. Each point represents a single chamber,
at different times after the introduction of varying numbers of viable Ehrlich ascites tumour
cells (n = 20, r = + 0-63.5, 0-001 < P < 0-01).

RESULTS

I. Proliferation of Ehrlich ascites tumour cells in diffusion chambers

This was investigated by quantitative determination of the population size at
different times after the introduction of various cell numbers (Table I).

Aliquots of 0-2 ml. Ringer solution containing 10^ to 10® viable Ehrlich ascites
tumour cells were pipetted into each chamber. The experiments with the smallest
inoculum (10^ in series 1, Table I) was repeated 6 times, while the other series
(series 2-5) are based on one experiment each. The total number of cells was
determined on the 20th and 30th day after implantation, with the exception of
series 1 and series 5 (cf. column 4, Table I). Only chambers contaming more
than 6x10^ cells were evaluated. About 50 per cent of the samples implanted
with 10^ cells were omitted since thej’^ contained fewer cells. The standard
deviation of the mean cell number is shown in column 7 in Table I. It is inversely
related to the inoculum size. A similar relationship was found for ascites tumours
gi'owing freely in the abdominal cavity (Kdein and Revesz, 1953).

The results are illustrated in Fig. 2. It can be seen that the tumour cells have
grown to multiples of the initial population size during the cultivation period.
The ratio of the mean cell number and the inoculum dose was inversely related to
the latter (column 8, Table I). This may be due to a gradual decrease of the
relative multiplication rate \nth increasing size as found with freely growing
ascites cells (Klein and Revesz, 1953). The largest population size, approximately

130

K. J. SEELIG ANE E. REVESZ

Table I. — Data from Diffusion Chamber Experiments with
Viable Ehrlich Ascites Tumour Cells

Series

number

Inoculum

Time

Number of

number of

after

separate

tumour

inoculation

experiment

cells

(da5's)

102

10*

102

i-sxio«

2, 6, 9, 12, 30

Emnberof R„,io

chambers of mean

used for Mean popuiation

separate population Standard size and
deter- size deviation inoculum
minations (log. units) (log. units) (log. units)

5 6 7 8

12(1)

5-28

0-650

3-28

15(2)

5-72

0-908

3-72

5-65

0-594

2-65

6-38

0-245

3-38

6-36

0-301

2-3G

6-46

0-406

2-46

6-52

0-257

1-52

6-77

0-239

1-77

6-91(2)

0-064(2)

0-91(2)

6-98(*)

—

o-g8(*)

0) Eleven chambers with less than 6 x 10^ cells are not included.

<“) Ten chambers with less than 0 X 10* cells are not included.

On the 12th day.

(*) On the 30th day.

Established from duplicate determinations at the intem'als indicated in column 4.

Growth of different doses of Ehrlich ascites tumour cells *" n!";;

I'fc cf.nnfinrf] fiFTOr is indicated. Each point represents the mean o o- o

Fig. 2.^-Kxiui^ vti ,

The mean and its standard error is indicated,

chambers.

effect of dai^iaged tumour cells on viable cells 131

9-5 X 10« cells per chamber, was attained after the

under the conditions used. Tliis corresponds to a concentration of about
cells per 0-1 ml. chamber fluid.

Fio. 3. — Percentage of celE with impaired nuclear stainability as related to population size
in the chamber. Differential counts were made on crj'Stalviolet stained material. The
upper diagram rejiresents chambers harvested 20 days after the introduction of 10^“ Ehrlich
ascites tumour cells (n = 7, r = 0-656, 0-05 < f < 0-1), while the lower diagram corres-
ponds to 30 days and 10* or 10^ inoculated cells (n = 12, r = 0-684, 0-01 < P < 0-02).

The results of differential countings on crystal violet stained material indicate
that the proportion of cells with far advanced nuclear damage increased with
time (Fig. 3). Wliile the great majority of the free Ehrlich ascites cells appear to
be viable during the entire growth period (Klein and Revesz, 1953), the percentage
of damaged cells increases in the chambers with time.

132

K. J. SEBLIGr AKD L. REVESZ

II. Behaviour of heavihj irradiated and lieat-ldlled cells in diffusion chambers

Two separate expCTiments were performed in order to study the viabilitv nf
heavily irradiated (HR) cells at vaiying intervals after introduction of lo' HR
cells suspended in 0-2 ml. Ringer solution. The eosin test of Schrek indicated
that the unstained fraction decreased progressive^ until after seven days the
overwhelming rnajority of the population became eosin stainable (Fitr. 4)
Occasionallj'^ eosin-unstained cells of considerably increased size were discernihle
as long as 16 days after incubation.

Fig. 4.— The percentage of eosin-iinstained cells in cliambers incubated with 10’ HR Ehrhrli
ascites cells. Each point represents a single determination in one chamber (tiro separat<5
experiments).

Disintegration of the HR cells proceeded at a slow rate. In 3 separate
experiments (series 6, Table II) using an HR inoculum of 5 X 10^ Ehrlich cel s
per 0-2 ml. about 90 per cent of the original population could be recovered on t le
15th day, and almost 40 per cent on the 30tli day (column 9, Table II). '' i®*'
suspended in citrate solution and stained with crystal violet, the recovered ce s
showed an indistinctly staining or completely dissolved nucleus. If
were incubated with 10^ HR cells for 150 days (series 7, Table II), about per
cent of the original inoculum were found in the fluid as diffusely stained ce u ar

entities. _ . , ,

In two separate experiments (series 8, Table II) the disintegration o i < ^
treated cells was followed. As the HR cells, the}’ disintegrated i

Approximately 70 and 50 per cent of an initial number of 5 X 10 hea
cells could still be identified after 15 and 30 days, respectively.

ettect op damaged tumour cells on viable cells

133

-.LE U.-Bata from Diffusion Chamber Experimaits with Viable andjor Treated Tumour Cells

Inoculum

Number Un-
of treated
separate Ehrlich
ries experi- ascites
Tiber ments cells

1 2 3

10 ^

10 '

Treated cells

TjTe

Treat-

ment

Ehrlich 12,000 r

Ehrlich Boiling
water bath
10 min.

MCIM

JISC

Liver

Ehrlich

12,000 r
12,000 r

12.000 r

Number

5 X 10'
5x10'

5x10'

5X10'
5 X 10'

10 '

5X10'

Ratio
of mean

Number of population

chambers

Time used for Mean

after separate population Standard untroaletl

' inoculation deter- size deviation inoculum

(daj’s) minations (log. units) (log. units) (log. units)

150

15
4

Boiling 5x10' 15

water hath 30

10 min.

(h Population size after substraction of initially admixed treated cells.

C-53

6-75

5- 79

6- 19

C-30

6-21

6-39

G-54

6-43

5-66

5-28

G-46

5-50

5-42

O-4G0

0-397

0-509

0-182

0-314

0-14G

0-263

0-211

0-194

0-146

4-53

4-75

3- 79

4- 19

4-30

4-21

4-39

4-54

4-43

III. Admixture of heavily irradiated tumour cells to a small viable inoculum

A large number (5 X 10^) of HR Ehrlich ascites tumour cells in 0-2 ml.
Ringer solution was admixed with 10^ viable cells in another 0-2 ml., and
introduced into the chamber (series 1, Table II). On the 15th and 30th day the
total cell number was determined in 25 and 14 chambers, respectively. Correction
was made for the initial number of treated cells by substracting it from the total
number. This was considered necessary since both irradiated and heat-killed
cells could maintain themselves in the chamber nearly quantitatively during the
observation period. Since some of them may have disintegrated, the correction
employed may have introduced a slight underestimation of the extent of growth.

A comparison between series 1 in Table I and series 1 in Table II indicates
that 10-20 times larger cell populations were attained in the presence of HR cells
than in their absence. This must be regarded as a minimum estimate since about
half of the control chambers inoculated with small viable inocula alone contained
less than G x 10* cells and therefore were eliminated from the calculations.
Even so the differences were highly significant (day 15 : P < 0-001 • dav 30 •
0-01 > P > 0-001). » O' -

Similar results were obtained when the lymphoma L1210 was grown in 0-4
ml. diffusion chambers, either in the presence or in the absence of HR cells of the
same kind. The inocula contained lOS 10* or 10« viable cells suspended either

134

K. J. SEELIG AND L. E^IVESZ

Tl solution alone or in 0-2 ml. Ringer containing 10^ HR cell.

HR opnff CorrecHon was made by substracting the number of added

HR cells from the figures. Pjg. 5 shows that considerably larger populations ivere
obtained in the presence of HR cells in this case too. The multiplication of tlie
Ij m^homa cells decreased with mcreasing population size and^ approached a
maximal value of about 150 x 10« total cells per chamber. Tins corresponds
to a concentration of about 36 x lO® cells per 0-1 ml.

Eig. 5. — Cell content of diffusion chambers after inoculation of 10^ 10* and 10' L1210
lymphoma cells either alone or together with 10^ HR cells of the same kind. The points
represent means of 2-6 determinations, and the range is indicated. The number of irradiated
cells was subtracted from the actual figures.

IV. Admixture, of heat-hilled tumour cells to a small viable inoculum

Ehrlich ascites tumour cells were suspended in Ringer’s solution to a con-
centration of 5 X 10® per 0-2 ml. and killed by immersion into boiling water for
10 minutes. This suspension was mixed in 0-2 ml. aliquots with another 0-2 ml.
Ringer solution containing 10^ viable Ehrlich cells and introduced into diffusion
chambers in the usual way (series 2, Table II). The cell contents of the chambers
were determined after 15 and 30 days. The initial number of heat-killed cells a as

substracted from the total number. i -ii I

Somewhat larger cell populations were attained in the presence of heat-kidca
cells than in their absence (series 1 in Table I and series 2, Table H)- ®

differences were not significant : P > 0-1 after 15 days, P ~0-3 after 3 ajs.

The difference between the HR group and the heat-killed cell admixture vas

135

effect of damaged tumour cells on viable cells

significant after 15 days (P < 0-001) but not after 30 days (P ~ 0-3) (series 1 and

2 in Table II).

V. Effect of foreign tumour or liver cells

To study the possible specificitj'^ of the stimulating effect, 10- viable Ehrlich
cells were introduced into the chamber together with 5 X 10® HPv cells derived
either from the J^IClAI ascites sarcoma or from the solid ^ISC fibrosarcoma (series

3 and 4, Table II). The size of the population was deternuned after 15 and 30
days, after subtracting the initial number of foreign irradiated cells. After lo
days the corrected cell numbers were about 10 times larger than in the controls
(series 1, Table I) (0-01 > P > 0-001). After 30 days the mean cell numbers were
still larger than the controls but the difference was not significant (P > 0-3).

In one experiment (series 5, Table II) 10^ Ehrlich cells were gronm in the
presence of approximately 5 X 10^ cell of an A.SW mouse liver suspension. On
the 15th day the size of the tumour cell populations was determined in 3 chambers
after differential counting tumour and liver cells. A stimulating effect was
observed, comparable to that observed with heavily irradiated Ehrlich cells.

DISCUSSION

The number of EhrUch ascites tumour cells increases at a slower rate inside
the diffusion chambers than in the peritoneal fluid. A comparison of the grondh
curve of an inoculum of 1-8 x 10® Ehrlich ascites tumour cells in the peritoneal
cavity (Klein and Pvevesz, 1953) and in the chambers shows a nearly logarithmic
correlation (Fig. 6). Whereas the percentage of eosin-stainable cells was neghgible
throughout the entire period of ascites growth in the peritoneal cavity (Klein
and Revesz, 1953) an increasing number of dead and degenerating cells could be
found in the chambers, however.

The differences between the intraperitoneal and intrachamber growth can be
assumed to be due to the fact that the diffusion barrier leads to a slower flow of
nutrients and an accumulation of waste products. The entrj^ of fluid into the
chambers has been studied by Amos and Wakefield (1958), They placed empty
chambers, corresponding in physical characteristics to those used in the present
investigation, into the peritoneal cavity of mice in order to study the rate of fluid
passage. After a lag of about 6 hours, filling proceeded at a rate of 0-085 ml.
per day and after 7 days the chambers were filled. When high titer isoantiserum
was injected intraperitoneally an equihbrium was obtained, usually after about
90 minutes, between the haemagglutinin levels of the peritoneal fluid and the
chamber contents.

It has been found that the growth of the Ehrhch ascites carcinoma in the
peritoneal fluid is characterized by a progressively decreasing multiplication rate,
coming to a stand-still after a maximum population had been reached. Quahta-
tively similar growth cunms were obtained in the chamber. The maximum cell
concentration of Ehrlich ascites tumour cells was about 1-3 x 10® cells per 0-1 ml
m the chamber -, tlus is approximately one tenth of the cell concentration in the
free ascites fluid (Klein and Revesz, 1953). With the L1210 lymphoma, however,
the concentration m the chamber, 36 x 10® ceUs was similar to the values found
wnth this tumour m the ascites fluid (Shelton and Rice, 19586). A third ascites
tumour, ELD, reached one third of its usual cell concentration in the ohaSber

136

K. J. SEELIG ANJD L. KEVBSZ

(11 X 10® cells per 0-1 ml.) (Norman, not yet published^ r):fr

(oTdva S considerably in their nisSS

consideiable proporfaon of the orjgjnal inoculum was distinctly discernible for
more than four weeks In her experiments with different lymphomas grovmin
diffusion chambers, Shelton found that soon after incubation dead cells became

•o

Fig. G. — Correlation between total cell number nt corresponding times in the peritoneal
fluid and in the chamber after inoculating 1 ■ 8 x 10' Ehrlich ascites tumour cells intra-
peritoneally or into the chamber, respectively. Regression line coefficient: 3-59.
Chambers of 0-8 ml. volume were used.

a large component of the population (Shelton and Rice, 1958a). It would seem
that the chamber fluid has little, if any, proteoljffic activity, perhaps due to the
absence of inflammatory cells.

In contrast to this situation, radiation damaged cells in direct contact with
host cells disintegrate soon after implantation. Inflammatory but no tumour
cells were found in the ascites fluid of mice 6 days after intraperitoneal injection o
Ehrlich ascites cells irradiated with 4000 r (Revesz, 1955). Genetically com-
patible sarcoma cells treated with 12000 r and injected subcutaneous^, uere
found embedded in a granulation tissue until the 7th day, but not later (Ringer z
et al, 1959). It would seem that disintegration of irradiated tumour cells at
either a subcutaneous or intraperitoneal implantation site occurs at abou le
same time that the majority of similarly treated cells introduced into chambers
become eosin-stamable (Fig. 4).

137

EFFECT OF DAjMAGED TUMOUR CELLS ON VIABLE CELLS

The stimulating effect of irradiated cells upon the groudli of admixed viable
cells as ohseiu^ed in the chambers is in conformity Avith analogous findings u'itn
ascites cells in the peritoneal fluid (Revesz, 1955 ; Scott, 1957 ; Mazurek and
Duplan, 1959) and with A’arious tumours in the subcutaneous tissue (Revesz,
1958) Two possible mechanisms have been considered previously to explain
the phenomenon ; a “ feeder ” effect (Puck d aL, 1956) based upon the release of
nutrients and/or growth stimulating substances or, alternatively, an effect
mediated tlirough local host responses. The possibility of a systemic host effect
Avas excluded since no stimulation was obtained when viable and heavily irradiated
(HR) cells were inoculated at two different anatomical sites (Revesz, 1958).
Histological studies of the implantation site actually revealed a certain correlation
between the ability of a given cell preparation to provoke an intense granulation
reaction and its stimulating effect (Ringertz d al., 1959). It was concluded that
the host reaction may be an important factor acting probably by enhancing
A^ascularization and stroma formation.

The chamber experiments show that HR cells can stimulate even in the
absence of any cellular host reaction. This is analogous AA'ith the finding that
single HeLa cells in vitro show an increased plating efficienc}’- and accelerated
groAvth rate if plated in the presence of a “ feeder layer ” of X-irradiated cells
(Puck d al., 1956).

The stimulating effect does not appear to be tumour specific since heaAuly
irradiated suspensions of foreign mouse tumours and a suspension of liAmr tissue
are also effectRe. This finding is in harmony Avith the preAuous obser\mtions
that groAvth of small subcutaneous inocula of different mammarj" carcinomas
and methylcholanthrene-induced sarcomas can be stimulated bj’^ admixed suspen-
sions prepared from lumr (ReA^esz, 1958), or embrj^onic tissue (Schnej^er, 1955 ;
VasilieA’^ and Olshevskaja, 1958) or from different tumours (fflein and IClein,
1956). It is at Amriance Avith the findings of Mazurek and Duplan (1959) Avho
maintain that stimulation is specific for the cells of the same tumour.

At the subcutaneous site heat-killed tumour cells exhibited no stimulating
effect (Revesz, 1958). In the chambers heal-treated cells showed a certain
stimulating activity which was, however, significantly less than that of radiation-
killed material.

srauLVRY

The influence of a large number of irreversibly damaged tumour cells upon
the subsequent proliferation of a small, admixed viable cell fraction was studied
in difiusion chambers inserted into the peritoneal caAuty of mice. A method is
described for quantitatiA-e estimation of the size of the cellular population in the
chambers. A considerable proportion of the original population of X-ray
inactivated or heat-killed cells was distinctly discernible in the chambers for more
than four weeks. Viable cells admixed to heavfiy irradiated material of the
same or different tumours or to liver tissue were found to grow more rapidly than
the same number of Auable cells cultured alone. Heat-killed cells enhanced the
groAvth to a significantly less extent.

The authors AAish to express their sincere gratitude to Dr. Ulla Xorman for
Aaluable suggestions regarding the methods used in this work This work has

138

K. J. SBELIG AND L . REVSEZ

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Amos, D. B. and Wakefield, J. D. — (1958) Ibid., 21, G57.

Klein, G. — (1951) Exp. Cell. Res., 2, 518.

Idem and IGlein, E. — (1956) Nature, 178, 1389.

Idem AND Revesz, L. — (1953) J. not. Cancer Inst., 14, 229,

Law, L. W., Dunn, T. B., Boyle, P. J. and Miller, J. H. — (1949) Ibid., 10, 179.
Mazdrek, C. and Duplan, J. F. — (1959) Bull. Ass.frang. Cancer, 46, 119.

Puck, T. T., Marcus, P. I. and Ciecidra, S. J. — (1956) J. exp. Med., 103, 273.
Revesz, L. — (1955) J. nat. Cancer Inst., 15, 1691. — (1958) Ibid., 20, 1157.

Ringertz, N., Klein, E. and Revesz, L. — (1959) Cancer, 12, 697.

ScHNEYER, G. A, — (1955) Cancer Bes., 15, 268.

ScHREK, R. — (1936) Amer. J. Cancer, 28, 389.

Scott, 0. C. A. — (1957) Brit. J. Cancer, 11, 130.

Shelton, E. and Rice, M. E. — (1958a) J. nat. Cancer Inst., 21, 137. — (19586) Ibid.,
21, 163.

Snell, G. D. — (1955) Transplant. Bull., 2, 6.

Vasiliev, Jd. and Olshevskaja, L. V. — (1958) Voprosy onkologii, 4, 548.

]39

EFFECT OF CHEMOTHEEAPEUTICS ON THE NUCLEIC ACID

ilETABOLISM OF TUMOURS

INCORPORATION OF INTO NUCLEIC ACIDS FOLLOWING
TREATMENT WITH DEGRANOL

E. J. HIDVISGI,* E. ANTONIt AND K. LAPIS

From the Institute of Biochemistry, Medical University, and the Research Institute of

Oncopathology, Budapest, Hungary

Received for publicntion November 18, 1059

Outstanding among tlie significant results acliieved in recent years in the
study of nucleic acids has been the recognition of tlie parallelism of their syn-
thesis to mitotic cellular acthdty (Hershey, 1954 ; Healy et al., 1956). Owing to
their supreme significance in the metabolism of tumours, and in tumour therapy
efforts are now concentrating on the development and utilization of agents which,
directly or indirectly, act upon them (Skipper and Bennett, 1958 ; Shive and
Skinner, 1958).

Chemotherapeutics, particularly those which act on the nucleic acids, are
nitrogen mustard and its derivatives. One of them is the compound, first
prepared by Vargha (1955) 1, 6-bis (/?-chloroethylamino)-l, 6-dideoxy-D-mannitol-
dihydrochloride, known as Degranol or BOM, the structural formula of which
may be represented as follows :

CH 2 -NH 2 -CH 2 -CH 2 -CI

HO-C-H

H-C-OH

CH 2 -NH 2 -CH 2 -CH 2 -CI

.vf tumour-inhibiting and haematological effects Degranol has been studied

xtensively by Kellner and co-workers (Kellner and Nemeth, 1956; Kellner 1956'

Nomcth, Kellner and Laps, 1958 ; Lapis and Nemeth, 1966a, 4) who Sd th!i:

140

E. J. HIDVEGI, F. ANTONI AND K. LAPIS

tlie morpholo^cal changes to which the drug gives rise foUow one another at
fairly regular intervals : mitoses begin to be damaged 6 to 12 hours after flip
administration of a smgJe dose ; the peak effect mvolving extensive destnictioii

the m^hoOT •'It about

On tJie analog of the earlier morphological investigations, we studied tlie
djmamics of the effect Degranol exerts on the nucleic acid metabolism of tumours,
a he present paper reports the results of experiments undertaken to shed lioht
changes which a smgle dose of the drug brings forth in the incorporation
of P into the nucleic acid fractions and acid-soluble nucleotides of transplanted
tumours.

JIATBRIALS AND METHODS

Experimental animals and their treatment tvith Degranol . — Inbred albino rats
weighing 120 to 150 g. were inoculated subcutaneouslj'^ with Guerin carcinoma
and 14 to 16 daji’s later given hitraimnouslj' a single 50 mg. /kg. dose of Degranol
Following this treatment thejf were decapitated at various times (24, 48, 72, 90
hours), but always after a 24-hour fast. The tumours they developed were re-
moved and found to iveigh from S to 12 g. Thin slices were cut off these tumours,
fixed in formalin and embedded in paraffin for histology. Eaeli tumour was
worked up separately, mostly using its florid parts only. Whole tumours,
how'ever, avere used in recovering ribonucleic acid (RNA) according to Pain and
Butler (1957).

The ^^P was applied in the form of K2HPO4, after hydrolysis (to eliminate
possible pyrophosphate) and neutralization. Each animal received a 100 ;ic/100
g. intraperitoneal dose of ^^P, 24 hours before decapitation.

Specific activity determination of acid-sohihle nucleotides . — ^Hecht and Potter’s
(1956) method was emploj^ed in extracting the acid-soluble compounds. Tumour
tissue (I g.) was broken up thoroughly in a Potter -Elvehjem tj'pe homogenizer
with 2 ml. of cold 0-6 n perchloric acid. The precipitate was centrifuged and
washed with 0-2 N HCIO4. The supernatants were pooled and neutralized with
KOH in the presence of phenol red as indicator. The KCIO4 was centrifuged off
and the tissue inorganic orthophosphate (Pj) was removed by treatment with
magnesia mixture (pH = 9) to which unlabelled phosphate had been added.
The acid-soluble nucleotides were then adsorbed from an acid medium on active
carbon and the adsorbate was centrifuged off, washed, and several times ehited
with 50 per cent ethanol containing 2 per cent NH3. The eluate was lyophilized
and the residue taken up in wmter. Portions of the solution were used for rac 10-
activity determination and for reading the nucleotide content in a Be^mann
model DU spectrophotometer at 260 m/( in a 1 cm. quartz cuvette, bpecitic
activity was expressed as cpm/1 Egeo ra,< (Bileri and Ledoux, 1957).

Measuring incorporation of into deoxyribonucleic acid {DNA), nuclear 1
{nRN A) and cytoplasmic RNA (cRNA ). — Using a Potter-Elvehjem tjqie homogeii
izer, the fragmented tumour tissue urns homogenized in ten times its volume 0
cold 0-25 M sucrose containing 0-005 M of CaGlg, and the homogenate was cen n
fuged in the cold at 600 g for 10 minutes, according to the method of Bogeboon ,
Schneider and Striebich (1952). Putting aside the cytoplasma supernatant, n
precipitate was twice w-ashed with 0-25 M sucrose, allowed to stand m - per c

INCORPORATION OF ^^P INTO NUCLEIC ACIDS

141

citric acid twice for half an hour each time, and washed twice with 2 per cent
acetic acid (Nygaard and Rusch, 1955). All washings and centrifugations were
done in the cold ; the latter invariably at 600 g for 10 minutes. n r

The nuclei isolated in the manner described were precipitated witli O-.j N
HClOj, and the cjdoplasmic fraction Avas brought to 0-55 N HClOj concentration
(Hurlbert and Potter, 1952). The precipitate was washed in the cold seven
times with 0-2 n HClO^ and twice with ethanol, whereafter it was e.xtracted three
times Avith a 3 : 1 mixture of ethanol and ether for 15 minutes at CO G. Ihe
residue Avas washed Avith ether and dried in vacuo. From the dry poAi'der the nucleic
acids AA'ere isolated by the method of DaAudson and Smellie (1952) . extraction
Avas performed Avith 10 per cent NaCl (pH = 7) for 60 minutes at 100° C., and the
procedure Avas repeated. The cRNA and iiRNA Avere hydrolyzed Avith 0-3 N
KOH for 1 6 hours at 30° C. In the nuclear fraction the DNA Avas separated from
the RNA nucleotides by acidification folioAving hydrolysis. The DNA V'as
immediately centrifuged off, dissolved in dilute NH4OH, purified by repeated
precipitation, and finally dissolved in dilute NH4OH.

Possible contaminating P; Avas removed from the RNA nucleotides by means
of magnesia mixture Avith unlabelled Pj added to it.

Portions of RNA and DNA fractions Avere used for radioactivity determinations
and for reading the nucleic acid contents. The RNA content Avas determined
by Mejbaum’s (1939) orcinol reaction as modified by C'eriotti (1955), and the
colour AA'^as read at 660 m/t on the Beckmann model DU spectrophotometer. The
DNA content Avas determined by Dische’s (1930) diphenylamine reaction as
modified by Seibert (1940) and the colour was read at 600 m// on the Beckmann
model DU spectrophotometer. Specific activity AA'as expressed as cpm/100 //g.
of RNA-P and DNA-P, respectively.

Relative specific activity. — ^Following Smellie et al. (1955), the specific activity
of the nucleotides and nucleic acids was referred to that of the Pj in the blood of
the experimental animal and stated as relatiAm specific activity, i.e. :

RelatiA^e specific activity

specific actiA’^ity of fraction studied
specific activity of blood Pj

X 10000

Determination of specific activity of in the blood. — The decapitated animal’s
blood Avas collected, and half its Amlume of trichloroacetic acid (20 per cent) v/as
added to it. The mixture Avas centrifuged, and from the supernatant the P; Avas
isolated in the cold AAdth magnesium mixture (pH = 9) as MgNH 4 P 04 . This Avas
recrj^stallized, and dissolved in dilute acid. Portions of the solution Avere then
used to determine Pj by the method of Fiske and SubbaroAV (1925), as modified by
Lohmann and Jendrassik (1926) and to measure radioactiAuty. Specific actiAuty
Avas expressed as cpm/100 /tg. of Pj ; as mentioned above, to this Avas referred the
specific activity of the nucleotides and nucleic acids, respectively, to be stated as
tlie relatiA'e specific actiruty.

Isolation of RNA for heterogeneity tests Avas performed by the method of
Pam and Butler (1957). The specific acthdty of the RNA so obtained Avas ex-
piessed as cpm/100 /ig. of ribose.

rawt-Portion of the solution to be studied was measured into an
alunuinum .hsh and there aiiowed to dry. Bodioaotivity was then mSsurS
under an end-wmdow GM tube, ,vith the aid of an Orion 1871 typreomto

142

E. J. HIDVEGI, F. ANTONI AND K. LAPIS

RESULTS

Earlier animal experiments (Antoni el al, 1958 : Antoni Vavphn •

1959) showed tliat for the isolation of E.NA from liver labelled m'th
a 24-honr circulation time was the ideal. Even with subcellular particles S
circulation time proved to be the best for labelling nucleic acids. Support
these findings lias been provided by the work of Tjmer, Heidelberger and LPa<re
(19o3) on tumours, for whicii, in turn, we have established confirmation in respect
of Guerin tumour. ^

Table I. — B/fecl of Degranol on Relative Specific Activity
of Tumour Nucleic Acids

100 pc of 3=P/100 g. of body weight injected 24 hours before decapitation.
Tlie figure.s in brackets indicate the number of animals involved

Relativo specific activity
Durnfioii of _jk

treutineiit

Control

DNA

3.030 (9)
3,530-4,200

nRNA
40,630 (10)
9,150-11,020

cRNA ’
S,30O (10)
6.950-8,750

24 hours

J.S50 (3)
1,410-2,460

7.530 (3)
7,100-9,820

S,050 (3)
7,050-8,250

4S hour.s

1,320 (4)
1,120-1,860

6,SJ0 (4)
6,170-7,830

7,230 (4)
6,810—/ /50

72 Iiours

E.\'perinient a

020 (7)
720-1,040

4,930 (7)
4,610-6,050

4,220 (7)
3,790-4,540

72 hours

E.vperimciit b

2,000 (3)
1,940-3,720

40,920 (3)
9,100-11,430

9,240 (3)
7,110-9,850

96 hours

4,420 (3)
3,840-4,960

44,230 (3)
10,620-12,700

S,S20 (3)

8 070-9,440

Table I illustrates the effect Degranol exerted in our experiments on the
incorporation of into tumourai nucleic acids. It shows that the drug caused
their relative specific activity to decline at rates wliich followed a certahi chrono-
logical regularity. The decrease was most marked in DNA ; more than 50 pp
cent in 24 hours and reachhig its minimum in 72 hours. It was less marked m
iiRNA and cRNA. At 96 hours after Degranol, i.e. at the time corresponding
to tlie begiiming of the restitutive stage as established b}’’ morphology,
relative specific activity^ of all the tliree nucleic acids studied was found^to ha\e
returned to the level seen in the controls. In some cases, as earty as i-
after treatment the relative specific activity'^ rvas of the same order of magni u e
in the experimental as in the control animals (cf. 72-hour experiment in
Table I). Histological examination showed the tumours of these animals o e
in the restitutive stage.

Since in the current view the acid-soluhle nucleotides are possible
of the nucleic acids our investigations were extended to the changes m
relative specific activity which follow treatment with De^anm. n ^
illustrates the efifect the drug had on the relative specific activity’^ of aci
nucleotides isolated simultaneously with nucleic acids and
carbon. The data reveal that similar dynamic forces were at work in s i p o

INCORPORATION OF INTO NUCLEIC ACIDS 143

the relative specific activity of both sources : the purified acid soluble nucleotides
and the nucleic acids. In some of our experiments the number of counts,
registered 72 hours after Degranol, was found closely to approach the limit of
errors ; when averaging experimental results such counts were taken into con-
sideration as non-ra^oactive values only.

Table II.— ^j5fec« of Degranol on Relative Specific Activity of
Acid-soluble Tumour Nucleotides

100 ftc of 32P/100 g. of body weight injected 24 hours before decapitation.
The figures in brackets inicate the number of animals involved

Duration of

Relative specific

treatment

activity

Control

3J0 (10)

262-340

24 hours

J50 (3)

120-194

48 hoims

S2 (4)

47-113

72 hours

20 (7)

Experiment a

0-73

72 hours

JOd (3)

Experiment 6

153-260

96 hours

320 (3)

290-365

It was possible for us to verify our results in RNA fractions isolated by a
different route (Pain and Butler, 1957).

The RNA was isolated from whole tumours, its specific activity was deter-
mined, and its heterogeneity studied (Antoni, Hidvegi and Lapis, 1960).

In Table III our experimental results are presented so as to demonstrate the
decrease in the rate of incorporation following treatment with Degranol ;
in other words, the specific activity of the RNA in the tumour of the treated

Table III. — Incorporation of^^P into RNA Isolated from
Whole Tumour According to Pain and Butler (1957)

100 fic of 32P/100 g. of body weight injected 24 hours before decapitation

Duration of
treatment
24 liours

Specific activity = control cpm/IOO jr g, of ribose
treated cpm/IOO pg. of ribose
1-86 (3)

1-30-2-15

”2 hours
9G hours

2-77 (6)
2-27-4-16

1-81 (3)
1-50-2-16

animal is referred to
These data show that

that in the tumour of the corresponding control animal
alterations induced by the drug in the rate of incorporation

144

E. J . HIDVEGI, F. ANTONI AND K. LAPIS

of 32p follow the same trend in RKA isolated by
from whole tumour.

a different method, namely,

DISCUSSION

Bodenstein (1 047) was the first to study the effect of nitrogen mustard on nucleic
acid metabolism. He found that the compound inhibited DHA sjmthesis, but
assumed it had no inhibitory effect on the sjmthesis of R,NA. (It would
appear that the drug has no such effect on nuclei during mitosis, but does e.xert
it in the interphase following mitosis.) Several authors hold similar views
(Lowrance and Carter, 1050 ; Skipper el al, 1951 ; Goldthwait, 1952). Harold
and Zippoiin recently found tliat mustard derivatives induced a transient inhibi-
tion of HHA symthesis in Escherichia cdli, but allowed ENA and protein symthesis
to continue unchanged.

Not unlike that of the other mustard derivatives, the initial effect of Degranol
(between 6 and 24 hours) is to damage mitoses and bring down the total miraberof
cell divisions. The peak effect falls between 48 and 72 hours, manifesting itself
in extensive cellular and nuclear disintegration in the florid parts of the tumour,
considerable expansion of necrotic areas, and the appearance of anomalous
mitoses (cacomitoses) and multinuclear tumour changes are followed by the
restitutive phase characterized by a cell population consisting predominantly of
giant cells (Kellner and Nemeth, 1956; Kellner, 1956; Nemeth, Kellner and
Lapis, 1958 ; Lapis and Nemeth, 1956n, 19565).

On the evidence of our experimental results treatment ivith Degranol is followed
by' a decrease in the incorporation of into the nucleic acids and acid-soluble
nucleotides of Guerin tumour. At the time it exerts its maximum effect the drug
reduces the level of incorporation into DNA to about a quarter of tlie original.
Incorporation into ENA and cRNA likewise shows decreases ; these however
are less marked.

Our results, further, permit the statement that the changes in nucleic acid
metabolism seen after Degranol are a dymamic process, the same as the morpho-
logical changes. In 24 hours a single dose decreases the relative specific activity
of DNA by' 50 per cent and reduces tlie total number of cell divisions. The drug
exerts its maximum effect betiveen the 48th and 7 2nd hour after input. Tliis is the
time when the morphological picture shows extensive cellular and nuclear disinfe-
gration and a progressive decline to the minimum of the relative specific actnity
of the nucleic acid fractions. This gradual decrease is particularly marked m
DNA, a phenomenon w'hich fairly' corresponds to the extensive nuclear disintegra
tion revealed by' moi’pliology'. Conformity w'ith the morphological
still more pronouned at 96 hours, ivhen the relative specific activity of J- >
iiRNA, and cRNA is observed to have returned to the level seen in the contj®' ’
coinciding with the appearance of multinuclear giant cells and
so great as to be practically' the only building stones of the entire

Conflicting interpretations of these cell forms are offered in the er
Many' authors have brought them into connection with regressive processes.
view', how'ever, is gaining ever more adlierents that they' are very'
interesting forms of cell division. Kellner’s studies (1952, 1960 ) also °
idea that these lands of cells have a decisive part to play in the restitu n e p
that follow' damages caused by' chemotherapeutics. By' our f rises

these cells come into prominence the level of relative specific activity o

145

IXCORPORATION OP ®-P IXTO NUCLEIC ACIDS

to that observed in the controls, n-hich phenomenon seems to confirm that their
appearance may actually be connected with processes progressive, and not
regressive.

While in some cases, at 72 hours after Degranol the relative specific activity
was the same in the experimental animals as in the controls, in others it showed the
low level which characterized the maximal effect of the drug. Such differences,
however, were mvariably found to conform ndth the changes established bj^
morphologjL For, whenever the relative specific activity was as high as in the
controls, the histological picture revealed changes tj^jical of the restitutive stage ;
conversely, whenever it was low, histologj’’ still showed marked nuclear destruc-
tion.

It seems safe to claim that our results offer the possibility of quantitatively
characterizing the alterations and their individual phases, to wliich the mustard
derivatives give rise in the tumour — and wliich hitherto could only be differentiated
qualitatively on the strength of morphological changes — ^l33’’ measuring the incor-
poration of into the nucleic acids.

SOtMARY

The compound 1, G-bis-f^-cliloroethjdammo)-!, e-dideoxj^-D-mamiitol-dihj^dro-
chloride (Degranol ; BCM) has been studied for its effects on the nucleic acid
metabolism of Guerin carcinoma transplanted mto rats. A single dose of the
drug was found to decrease incorporation of into the various nucleic acid
fractions. At 72 hours after injection the decrease was most marked ui DNA.
It was less marked in nRNA and cRXA. At 96 hours, ®^P incorporation into aU
the tliree nucleic acids studied was seen to have returned to the levels observed
in the control animals. Variations of a similar trend were noted in the acid-
soluble nucleotide fractions isolated simultaneouslj'^ with the nucleic acids.

The conclusion is drawn that the changes occurring in the nucleic metabolism
of tumours represent a djnamic process, as do the morphological changes.

REFERENCES

Axtoxi, F., Hidvegi, E. J. axd Lapis, K. — (1960) Nature, Land., 186, 81.

Idem, JosEPo^^TS, G., Hidvegi, E. J. axd Szekessy-Hekmax-x', V. — (1958) 4lA Iiit.
Congr. Biocliem., Vienna.

Idem, I'AEGHA, L. axd BAdvegi, E. .J. — (1959) Acta 'physiol. Acad. Sci. Ining., 16, 1
Bodexsteix-, D.— (1947) J. exp. Zool., 104. 311.

C'ERiom, G.— (1955) J. biol. Chem., 214, 59.

DA^^Dsox, J. X. AXD S-MELLiE, R. M. S. — (1952) Biochem. J., 52, 594
Dische, Z.— (1930) Microchemie, 8, 4.

Fiske, C. H. AXD StJBBAKOw, Y. — (1925) J. biol. Chem.. 66. 375.

Goldthwait, D. A.— (1952) Proc. Soc. exp. Biol. A’.F., 80, 503.

Mealy G. JL, SnnxoAUTCH, L., Parker, R. C. axd Graha^i. A. F.— (I956) Biochim
Bwphys. Acta, 20, 425.

Hecht, L. I. axd Potter, V. R. — (1956) Cancer Res., 16, 988.

Hershev. a. D. — (1954) , 1 . gen. Physiol.. 38. 145.

HoGEBooM.ia H., SCHXEIDER, W. C. AXD Striebich, M. J.-(1952) J. biol. Chem.,

Hvrlbert, R. B. axd Potter, V. R. — (1952) Ibid.. 195, 257
12

146

K. J. HIDVEGI, F. ANTONI AND K. LAPIS

Kellner, B.— (1952) 3ITA Biol, es Orv. Tvd. Osztdlydnah Kdzlememjei (Traimclions
of the Biological and Medical Section of the Hungarian Academy of Sciences—
Himgarian only), 7, 435.— {I960) Acta Un. hit. Cancr., 16, 80.— (1956) Acta
morph, hung., 7, 215.

Idem AND Nemeth, L. — (1956) Z. Krebsforsch., 61, 165.

Lapis, K. and Nemeth, L.— (195Cf/) Klin. Wschr., 34, 864.— (19561)) Brit. J Dancer
10, 719.

Lohjlvn, K. and Jendrassik, L. — (1926) Biochem. Z., 178, 419.

Lom-rance, P. B. and C.VRTER, C. E.— (1950) J. cell comp. Physiol, 35, 387.

aiEJBAUJi, W.— (1939) Z. physiol. Ghem., 258, 117.

Nemeth, L., Kellner, B. and Lapis, K. — (1958) Ann. N.Y. Acad. Sci., 68, 879.

NYGAiVUD, 0. AND Ruscii, H. P. — (1955) Cancer Res., 15, 240.

Pain, B. H. and Butler, J. A. V. — (1957) Biochem. J., 66, 299.

PiLERi, A. AND Ledoux, L. — (1957) Biochim. Biophys. Acta, 26, 309.

Seibert, P, B. — (1940) biol. Chem., 133, 593.

Shive, W. AND SiaNNER, G. G. — (1958) Annu. Rev. Biochem., 27, 643.

Skipper, H. E. and Bennett, L. L. Jr. — (1958) Ibid., 27, 137.

Idem, JiliTCHELL, J. H. Jr., Bennett, L. L. Jr., Newton, M. A., Sebpson, L. andEiDSON,
31. — (2951) Cancer Res., 11, 145.

Smellie, R. 31. S., Humphrey, G. F., KAy, E. R. 31. and DA^^DS0N, J. N.— (1955)
Biochem. J., 60, 177.

Tyner, E. P., Heidelberger, C. and Lepage, G. A. — (1953) Cancer Res., 13, 186.

Vargha, L. — (1955) Naturwissenschaften, 42, 582.

147

INDUCTION OF TUI\IOURS BY TANNIN EXTRACTS

K. S. KIRBY*

From the Chester Beatty Research Institute, Institute of Cancer Research
Royal Cancer Hospital, London S. TF.S

Eeceived for publication February 1, 1960

Tafnic acid was shown to be carcinogenic to rats by Korpassy and Mosonyi
(1950) and to have a synergistic effect with acetyiamino-fluorene on the production
of liver tumours in rats (Mosonyi and Korpassy, 1953 ; Korpassy, 1959).

Tannic acid is only one of a group of naturally occurring compounds which are
knoAvn as vegetable tannins and are in general polyphenolic substances which have
the ability to form a precipitate with gelatin under prescribed conditions. All
these tannin extracts are mixtures of a number of components and no two are
identical (White, Kir by, Kmowles, 1952) although some compounds (e.g. gallic
acid, ellagic acid, catechin) may be common to a number of extracts.

Some of these tannin extracts have been tested against both rats and mice
■with a view to determining the range of carcinogenic activity of this group of
substances.

MATEKIALS AND METHODS

Animals. — ^Rats of the August strain and stock and C 57 black mice were used
for the experiments.

Tannins . — The following extracts were used : sidphited Quebracho (Schinopsis
lorentzii), kCmosa {Acacia moUissima), Myrtan (Eucalyptus redunca). Chestnut
(Castanea saliva Mill.), Myrobalans (Terminalia chebula Retz), Valonea (Quercus
aegilops L.) and Tannic Acid (B.D.H.). A single compound, fraction I from
quebracho extract (Karby, Knowles and White, 1953) has also been investigated.
This substance was insoluble in water and was injected as a solution in 50 per cent
propylene-glycol-water. In addition pure catechin and gaUic acid have been
employed. Tannin extracts usually contain 10-15 per cent moisture and 600 mg.
of the extract was dissolved in water, adjusted to pH 6-6-5 with n /10 NaOH
and the whole made up to 100 ml. with water. The solution was filtered if
necessary before using and stored in a refrigerator. Rats were given a subcu-
taneous injection of 1 ml. each per week for 12 weeks and mice were given 0-25
ml. each per week for 12 weeks.

Feeding experiments. — Mice were allowed to drink 0-1 per cent solution of
sulphited quebracho and of myrtan extracts for 3 months and then 0-5 per cent
solutions of the same materials for 3 months. Jlice drank these solutions in place
of water and in fact, drank somewhat greater volume of the tannin solutions than
other mice draiik of water during the same period.

* Britisli Empire Cancer Campaign Fellow.

12 §

14S

K. S. KIRBY

RESULTS

Groups of 10 August rats were eacJi injected R-ith solutions of sulphitert
quebracJio, nuniosa, injTobaJaJis arid taiutic acid. Sarcomas at the site of iniec
tion appeared about 1 year iater in 2 August rats in each group of tiiose injected
witdi suljjliited quebraeho and mimosa extracts. Some of the animals in these
group.s had been killed earlier to look for damage to the liver, but none was
observed, lioth tumours produced by these extracts have been transplanted and
have noAV reached the poth generation. The original transplants required about
6 wcclts to grow to a size of about 2 cm. diameter but after about 5 generations
this period was reduced to 12 days. Tannic acid and mjTobalan extracts had no
observable effects on August rats.

dlice. — A wider group of tannin extracts lias been used upon mice and the
results are summarized in Table I.

Table I. — Groiiji of Tannin Extracts Used Upon Mice

Extract.

Stock mice —

C<, mice —

Jlyrtan
Quebrnolio
Miinosft
Tannic acid
Jtyrobalans
Chestnut .
Vnlonea .

Quobrnelio (40)
Oiostnut (20)

Sarcomas

Liver tumours

Some Jjunphomas were also observed in these groups but they are of little
.significance, since they occur spontaneously in older stock mice.

Feeding experiments. — 0-1 per cent solution of sulphited quebracho and of
mjTtan in water (pH not adjusted and was about 4-5) were fed to mice during 3
months and then 0-5 per cent solutions were fed for a further 3 months, ko
adverse effects were noticed in any of the animals during 1 year.

Histology. — ^hlicro-photographs of sections of some of the tumours obtaine
bj'' injecting tannin extracts into rats and mice are shown in Tig. 1-8-
show different tjrpes of sarcomata induced by quebracho, sulphited quelracho an
mimosa extracts and Tig. 5-S show liver tumours induced by myrtan and ches nu
extracts and sarcomata by m^mtan extract.

DISCUSSION

The effects produced in these experiments have been with minimum
of the tannin extracts. Korpassy and Mosonyi (1950) produced hepatoma ^
up to 9950 mg, of tannic acid/kg. body weight by injection, wlfile m le e. p
ments described a carcinogenic effect was observed after a total oi o &•
extract/kg. bad been injected mto the rat and 750 mg./kg. into the • j...

The effect is not due to ulceration which was observed in some pre i .
injections with tannic extracts and the dose was lowered so that no
ulceration was observed. Chestnut tannin extract was by far the most a o o

nsTDUCTIOK OF TUMOUKS BY TANKIN EXTRACTS

149

in this respect but no sarcomata resulted from injection of this material. While
only a small number of tannin extracts have been investigated a correlation can
he noted betweeii the tumour resulting and the type of extract injected.

Tannins , may be classified quite generally’’ into 2 tjqies ; (1) hydrolj'^sable
tannins which on heating with mineral acids jneld glucose, possibly other sugars
and gallic and related poljqohenolic acids ; (2) condensed tannins which produce
intractable red precipitates on heating with mineral acids. While little of the
chemical nature of these latter type of tannins is knovm, poljqihenolic residues
are present and may be linked tlirough polyhydroxjdlavan nuclei.

Of the tannins used in this investigation quebracho, mimosa and m3ntan
extracts are of the condensed variety wliile mjTobalans, chestnut and valonea
extracts are hydrolysable tannins. While all the extracts induced some liver
tumours, only the condensed tannins produced sarcomata at the site of injection.
Moreover, when 2 condensed tannins were fed tlirough the drinking water no
lesions were observed, in agreement ivith the results of Korpassy and Mosonyi
(1950) who observed no liver tumours by feeding tannic acid.

Although tannins are complex mixtures it was found possible to induce a
sarcoma in the mouse by injection of a single component (judged by a paper
chromatography) from quebracho extract and possiblj’^ only a small number of
the components present in any one extract may be carcinogenic.

The production of a sarcoma at the site of injection by condensed taimins is in
agreement with the experiments of Armstrong, Clarke and Cotclun (1957) who
suggested that condensed tannins were held at the site of injection more firmly
than hydrolysable tannins.

Gallic acid has been reported not to have any carcinogenic activity and
catechin (3 : 5 : 7 : 3' : 4'-penta-hydroxyflavan, the basis of some of the tea
tannins) is negative in mice at the levels tried in these experiments.

It is diffieult to speculate on the carcinogenic activity of tannin extracts
particularly when relatively little is known of their chemical structure and when
substances of such a wide difference of structure as 4-dimeth3daniino-azo-benzene
(Kinosita, 1937), thioacetamide (Rather, 1951), dimethjdnitrosamine (Magee
and Barnes, 1956) and ethionine (Farber, 1956) all produce liver tumours in the
rat or the mouse. However, the two important features of tannin reactivity
which are known are the reaction noth proteins wliich results in the formation of
leather and the ability to complex with metals, of which the complex between
tannic acid and iron salts is the most well known. Whether either or both of
these reactions are of importance in the carcinogenic activity must remain for
future experiments.

SUAEUARY

Tumours have been induced in rats and mice by subcutaneous injections of
various tannin extracts. Condensed tannins evoked sarcomata at the site of
injection as well as liver tumours, but liver tumours only were produced by
extracts of hydrolysable tannins.

This work was carried out during the tenure of a British Empire Cancer
Campaign Research Fellowship.

I vash to thank Professor A. Haddow, F.R.S. for his help and advice, the late
1 rofessor E. Homing and Dr. L. Foulds for the pathology, jMr. R. T. Charles for

K. S, KIRBY

RESULTS

Groups of 10 August rats %vere eacli injected u'ith solutions of sulnhited
qucbraclio, nuraosa, mjTobalans and tannic acid. Sarcomas at the site of iniee
tion appeared about 1 jmar later in 2 August rats in each group of tliose injected
until su phifced quebraclio and mimosa extracts. Some of the animals in these
groups had been killed earlier to look for damage to the liver, hut none was
observed. Both tumours produced by these extracts have been transplanted and
have nou' reached the OOth generation. The original transplants required about
6 u’eeks to grow to a size of about 2 cm. diameter but after about 5 generations
this period was reduced to 12 daj^s. Tannic acid and myrobalan extracts had no
obseiu'able effects on August rats.

Mice. A wider group of tannin extracts has been used upon mice and the
results are summarized iu Table I.

Table I. — Grov2> of Tannin Extracts Used Upon iJ/ice

Extract

SlocI: mice —

Ulyrtaii

Quebracho

Jliruosa

Tannic acid

Myrobnlans

Cliestnut .

Valonea

Cj, mice —

Quebraclio (40)
Ciiestnut (20)

Sarcomas

Liver tumours

. 5

. 7

Some l 3 ''raphomas were also observed in these groups but they are of little
.significance, since thej'^ occur spontaneously in older stock mice.

Feeding experiments. — 0-1 per cent solution of sulpliited quebracho and of
mjTtan in water {pH not adjusted and was about 4-5) were fed to mice during 3
months and then 0-5 per cent solutions were fed for a further 3 months. No
adverse effects were noticed in anj^ of the animals during 1 year.

Histology. — hlicro-photographs of sections of some of the tumours obtained
bj'^ injecting tannin extracts into rats and mice are shown in Tig. 1-8. Tig- l-^
show different tjqoes of sarcomata induced by quebracho, sulphited quebracho and
mimosa extracts and Tig. 5-8 show liver tumours induced by mjTtan and chestnu
extracts and sarcomata by mjTtan extract.

DISCUSSION

The effects produced in these experiments have been with minimum amounts
of the tannin extracts. Korpassy and Mosonyi (1950) produced hepatomata
up to 9950 mg. of tannic acid/kg. body weight by injection, while in the ,
ments described a carcinogenic effect was obser^md after a total of 35 mg-
extract/kg. had been injected into the rat and 750 mg./kg. into the mouse.

The effect is not due to ulceration which was observed in some prelimina >
injections with tannic extracts and the dose was lowered so that no ^

ulceration was observed. Chestnut tannin extract was by far the most a o

INDUCTION OF TUMOUBS BY TANNIN EXTEACTS

149

in this respect but no sarcomata resulted from injection of this material. While
only a small number of tannin extracts have been investigated a correlation can
be noted between the tumour resulting and the tj^je of extract injected.

Tannins may be classified quite generally into 2 tj’pes : (1) hydrolysable
tannins which on heating vith mineral acids yield glucose, possibly other sugars
and gallic and related polyphenolic acids ; (2) condensed tannins which produce
intractable red precipitates on heating with mineral acids. Wliile httle of the
chemical nature of these latter tjqje of tannins is knovm, pol5q3henolic residues
are present and may be linked through polyhydroxyflavan nuclei.

Of the tannins used in this investigation quebracho, mimosa and mjTtan
extracts are of the condensed variety while mjT’ohalans, chestnut and valonea
extracts are hydrolysable tamiins. While all the extracts induced some hver
tumours, only the condensed tannins produced sarcomata at the site of injection.
Moreover, when 2 condensed tannins were fed through the drinking water no
lesions were observed, in agreement until the results of Korpassy and Mosonjd
(1950) who obsenmd no liver tumours by feeding tannic acid.

Although tannins are complex mixtures it was found possible to induce a
sarcoma in the mouse by injection of a single component (judged by a paper
chromatography) from quebracho extract and possibly only a small number of
the components present in any one extract may be carcinogenic.

The production of a sarcoma at the site of injection by condensed tannins is in
agreement with the experiments of Armstrong, Clarke and Cotchin (1957) who
suggested that condensed tannins were held at the site of injection more firmly
than hydrolysable tannins.

Gallic acid has been reported not to have any carcinogenic activity and
catechin (3 : 5 ; 7 ; 3' : 4'-penta-hydroxyflavan, the basis of some of the tea
tannins) is negative in mice at the levels tried in these experiments.

It is difficult to speculate on the carcinogenic activity of tannin extracts
particularly when relatively little is knoum of their chemical structure and when
substances of such a wide difference of structure as 4-dimethylamino-azo-henzene
(Kinosita, 1937), thioacetamide (Rather, 1951), dimethylnitrosamine (Magee
and Barnes, 1956) and ethionine (Farber, 1956) all produce liver tumours in tiie
rat or the mouse. However, the two important features of tannin reactivity
which are knoum are the reaction with proteins which results in the formation of
leather and the ability to complex with metals, of which the complex between
tannic acid and iron salts is the most well known. IWiether either or both of
these reactions are of importance in the carcinogenic activity must remain for
future experiments.

SUJCMARY

This work was carried out during the tenure of a British Empire Cancer
Campaign Research Fellowship. ^ uaiicer

I wish to Riank Professor A. Haddow, F.R.S. for lus help and advice the late
rofessor E. Horning and Dr. L. Foulds for the pathologj^ I\Ir. R. T. Charles for

150

K. S. KIRBY

assistance wth the animal experiments, Mr. S. R. Scarfe for the preparation of
the histological sections, Jlr. K. Moreman for the photographs and Miss G, E,
Adams for technical assistance. The tannin extracts were kindly provided bv
Dr. T. IVhite and Dr. H. J. C. King of the Forestal Laboratories, Harpendeii
This investigation has been supported by’- grants to the Chester Beatty Eesearcli
Institute (Institute Ox Cancer Research ; Roy’^al Cancer Hospital) from the Medical
Research Council, the British Empire Cancer Campaign, the Jane Coffin Cliilds
]\Iemorial Fund for Medical Research, the Anna Fuller Fund, and the National
Cancer Institute of the National Institutes of Health, U.S. Public Health Service.

REFERENCES

Armstrong, D. M. G., Clarke, E. G. C. and Cotchin, E.— (1957) J. Pham., Loud.. 9. 9S,
Faeber, E. — (1956) Cancer lies., 16, 142.

ICtnosita, R. — (1937) Trans. Jap. path. Sac., 27, 665.

ICmny, K. S., Knowles, E. .vnd White, T.— (1953) J. Soc. Leath. Tr. Ghem., 37, 283.
Korpassy, B. — (1959) Cancer Res., 19, 501.

Idem AND Mosonyi, M.— (1950) Brif. J. Cancer, 4, 411.

Magee, P. N. and Barnes, J. M.~(1956) Ibid., 10, 114.

Mosonyi, M. and KorpAssy, B.— (1953) Nature, Land., 171, 791.

Rather, L. J. — (1951) Johns Hoph. Hasp. Bull., 88, 38.

White, T., Kirby, K. S. and Knowles, E. — (1952), J. Soc. Leath. Tr. Chem., 36, 148.

EXPLANATION OF PLATES
All sections were stained with hneinntox 5 lin and eosin.

Fio. 1. — Fibrosarcoma in the mouse induced by injecting a single component from quebracho
extract. This tumour shows a tendency to differentiate in the direction of the fibrous
connective tissue. { x 130.)

Fig. 2. — Spindle coll sarcoma in the mouse, induced by injecting sulphited quebracho e.vtraei.

Fig. 3. — Spindle cel! sarcoma in the rat induced by injecting mimosa extract. This material
was from a transplant of the loth generation. (Xl50.) , u f

Fig. 4. — Pleomorphic sarcoma in the rat induced by injecting sulphited quebracho ex r
Many giant cells are present. (xI30.) ... / luni

Fig. 5. — Early hepatoma in tlie mouse induced by injecting mjTtan extract. ( X *

Flo. C. — Hepatoma in the mouse induced by injecting chestnut tannin extract. ( X 1» •)

Fig. 7. — Mixed cell sarcoma in the mouse induced bj’ injecting mjTtan extract. 1 , , A

Fig. 8. — Spindle cell sarcoma in the mouse induced by injecting mjTtan extract. ( .x o .)

BlilTlSlt .loiIUNAL OK CaNCKTI.

Vol. XIV, No. 1.

Kirby.

British Jouun'ai. ok Canxhu.

Vol. XIV, Xo. 1.

Kirby.

BRITISH JOURNAL OF CANCER

YOL. XIY

JUNE, 1960

NO. 2

DE^^5LOP]\IENT OF CARCINOIMA OF THE CERVIX UTERI
Observatioks Resultusg fro:m Cyt'ological Exaimixation of
10,000 Cervical Sme.vrs

V. JI. BODDINGTON, B. H. COWBELL a>'d A. I. SPRIGGS
From the Deparhnenl of Pathology, United Oxford Hospitals.

Received for publication JMnrcIi 28, lOGO

Ix 192S Papanicolaou in New York and Babes in Bucharest independently-
discovered that carcinoma of the uterus sheds identifiable cells into the vaginal
fluid. This finding was later developed b}’- Papanicolaou and others into an impor-
tant diagnostic method v'hich has now been widely adopted. The cytological
diagnosis of carcinoma of the uterus has accumulated an enormous literature,
quite out of proportion to the limited information wliich can be extracted from the
examination of vaginal or cervical smears.

It soon became clear that the new method reveals certain abnormalities of the
cervical epithelium other than invasive squamous carcinoma. There is, in fact, a
whole range of cytological changes corresponding to the histological appearances
labelled “ at;jq)ical hyperplasia ” and “ carcinoma in situ ”, and the interpretation
of smears immediately became involved in the controversial problem of precan-
cerous change (Ayre, 1952a). Since the histological diagnosis is the universally
accepted yardstick by which the accuracy of cytological diagnosis is assessed, -vs^e
have the situation of the blind leading the blind.

The results of experimental work on carcinogenesis show that invasive cancer
develops in the course of a series of cellular transformations which are probably
discontinuous. After invasion has begun, further transformations may continue
to occur, producing increases in malignancy (Foulds, 1958). Among other sites,
this discontinuous transformation has been demonstrated experimentally in the
cendx uteri of the mouse (von Haam and Scarpelli, 1955 ; Reagan and Wentz,
1959). Ad the evidence from human material supports the same concept. In one
cer\nx a series of lesions may be found from basal cell hyperplasia or squamous
metaplasia up to invasive carcinoma (Howard, Erickson and Stoddard, 1951 ;
Gusberg and Moore, 1953 ; Carson and Gall, 1954).

Direct evidence of the precancerous nature of “ carcinoma in situ ” in the
uman cervix is of two kinds. In the retrospective study of Galvin, Jones and Te
inde (1952), out of 13 patients with carcinoma of the cervix who had undergone
lopsj^ one to seventeen years before, a re-view of the old material revealed “ carci-
noma m situ ” in 11. Many prospective studies have sho-«Ti progression of “ carci-
noma in situ ” to invasive carcinoma, often after a lapse of many years (Younge,
ertig and Armstrong, 1949 ; Gusberg and Moore, 1953) ; but this progression
13

152

M. 51. BODDINGTON, R. H. COWBELL AND A. I. SPRIGGS

has not occurred in every case. Unless regression is explained by total removal of
tile lesion at biopsy, it seems that we are not necessarily dealing with carcinoma
at all but with an epithelium which has climbed some of the steps towards malig-
nancy. The data of Petersen (1955; 1959, personal communication) show the
proportion of 127 untreated cases which progressed eventually to invasive carci-
noma. The cumulative percentage of cases in ivhicli carcinoma had developed
increased ivith each year that passed, at first rapidly, then more slowly, and after
7-S years the risk had become quite small. By extrapolation of the curve it can
be deduced that about 35 per cent of cases of “ cervical precancerosis ” (diagnosed
by Petersen) wdll sooner or later develop carcinoma of the cervix. “ Regression ”
usually occurred in the first year, and 50 per cent of those with lesions persisting
bej'ond this time developed carcinoma within ten years. Whether early invasive
carcinoma was present, but not found, at the time of the first biopsy in some of the
cases, it is impossible to decide. We also do not Imow whether the epithelium
which developed into carcinoma looked the same as the piece removed at biops}'.
These considerations do not invalidate Petersen’s figures from the point of view of
clinical significance.

If we accept that in only one-third of the cases mIU “carcinoma insilu” progress
to invasion, it is important to find out whether the cases can be sorted beforehand
into moqihological groups canying different prognoses. The patients are usually
3 ’’oung, and many wish for children. Also, if they are all treated as for carcinoma,
some niaj^ be left ivith serious comph'cations of treatment (Latour, Bromi and
Turnbull, 1957). With this in mind we decided to re-examine our material and
correlate as far as possible the cytologieal and histological appearances with the
clinical course.

We have also reviewed all the slides from patients whose cervical lesions have
been left untreated for a jmar or more. It seems very important to know whether
the abnormal cell-tjqie usually'’ remains the same over long periods, or whether it
alters to a more anaplastic type ; and if this happens, ivhetlier it happens suddenly
or graduallj'. Data on this point are extremely'’ scarce (Ayre, 1952a, 6) and our
series provides evidence about the stabihty'^ of the cell-ty'pe in ten cases.

PRESENT mVESTIGATJOX

BetM'cen 1952 and 1959 10,000 cervical smears were examined from 8522 cases,
most of which were gymaecological outpatients. The smears were taken by t e
examining gymaecologist by scraping the entire squamo-columnar junction area
with a ’ivooden spatula as described by Ayre {1952a), and smearing the
on a slide, and one slide w'as made on each occasion. The shdes were ^
ether-alcohol or 3 per cent acetic acid in ethyl alcohol, and they were stame y

the Papanicolaou method. kJoucv

The histological material available has usually been a ring or mulwpie p y
of the squamo-columnar region of the cen'ix. Sometimes a cone biopsyL
amputated cervix or the whole uterus has been submitted. Whenever
all the cervical tissue available has been embedded and either serial sec lo
multiple step sections examined.

During the period of 7 years, 11 cases of clinicaUy unsuspected mvasi
carcinoma of the cervix were detected, as well as 67 cases vdth possib y P ,
cerous cervical abnormalities. Of the latter group, 56 were clinically unsuspecw •

DEVELOPMEXT OF CARCINOMA OF THE CERVIX

153

Li the same period there were 9 adequate smears which were " false negative ’’
for cervical carcinoma, and 14 “ false positive ” in which histological studies
revealed no cause for the abnormal cells. In some of these cases, it is possible that
the area from which the abnormal cells were derived has not yet been removetl for
examination.

By reviewing the cytological and histological slides from all these cases we hoped
to answer the following questions ;

1. Can the histological findings be pretlicted from the cytological appearances?

2. If mvasive carcinoma develops from any of the other lesions, can tliis be
predicted either histologically or cytologieally? \Adiich txqjcs. if any, regress and
which persist unchanged?

3. Is the cjdological type consistent over a period of time, or does it change
cither gradually or suddenly ?

CLASSIFICATION AND TERMINOLOGY

A system of numerical histological and cxdological gi-ading was devised in
order to avoid any built-in opinion about the significance of the finding or its
prognosis. All the histological slides were reviewed and graded by the same
observer (R. H. C.) withouf knowledge of the clinical or c\*tological findings. The
ecological preparations were independently reviewed and graded by two observers
(M. M. B. and A. I. S.), and for this purpose the identification marks of the slides
were covered.

A. Histological Classificafiort

For the purpose of this investigation we have reviewed all the relevant slides
and classified them according to the following munerical system.

N^on-invasive lesions

1. Simple basal cell hyperplasia, occurring beneath either squamous or endo-
cervical columnar epithelium (Fig. 1).

2. A thick stratified squamous epithelium with hyjierkeratosis. JIany of the
superficial squames are nucleated and there is variation in size of these nuclei,
some being quite large, but the general epithelial pattern is orderly and mitoses
are few. The distinction from simple hyperkeratosis is based on the somewhat
^ emicous variation in the width of the epithelium and the variable size of nuclei
m keratinised cells (Fig. 2 ).

3. Sunilar to 2 with equally well-defined stratification but more mitotic
activity and nuclear irregularity (Fig. 3).

^"Pithehal activity at level of 3 but without the marked hvperkeratosis
(Fig. 4). ‘ • ^

5- Stratified squamous epithelium, often with considerable variation from
normal in the thickness of the strata liut with all represented. Mitotic activity

greater then 4 and moderate numbers of cells in aU lavers with larse hvperchroma-
tic nuclei (Fig. 5). - - -

n-itb shoiving some stratification, but poorly developed compared

tlnn L squamous epithehmn. Mitoses and abnormal cells numerous. A

"eratimsed lax-er present on the surface (Fig. 6).

154

M. M. BODDINGTON-, R. H. COWDELL AKd a. I. SPRIGGS

7. Stratification virtually absent but still sliaht sunerficial IrAmH-nJeof ■
some cases. Many mitoses iml abnormal cells kerahmsstan ,

Invasive carcinoma

differentiation in vdiich the tumour
cells tend to lie ni irregularly shaped solid aggregates vdth increasing maturity
towards the centre, where Iceratinisation may be present (Fig 8)

9. Squamous carcinoma including sheets of strikingly uniform large polygonal
cells, similar to prickle cells although usually without intercellular bridges (Fi^. 9).

10. Squamous carcinoma vuth a high proportion of grossly abnormal large cells
including giant cells vuth massive irregular nuclei and numerous mitoses, many of
them at 3 qiical. These tumours present an appearance similar to that seen in a
squamous carcinoma soon after irradiation, but the biopsies given this classifica-
tion were taken before radiotherapy’’ (Fig. 10).

11. A transitional ” ty'pe of tumour with broad bands of cells rather than
clumps. The general appearance is of fairly uniform hyperchromatism with no
Iceratinisation, and the individual bands of tumour cells are somewhat reminiscent
of the epithelium of a “ carcinoma m situ ” (Fig. 11).

Some explanation is required for the choice of the histological classification.
As regards the non-invasive lesions, so many names have been applied by different
authors that is has become the fasliion to quote a sample of them, discard them
and start afresli. Hinselmann (1953), in explaining the reason for formulating his
rubrics (Hinselmann, 1928), said that in his grading he consciously avoided contro-
versial terms such as pre-cancerosis and pre-invasive carcinoma, only aiming at a
more accurate definition of the existhig epithelial atypism. Our motives are similar
to his, and the present numerical classification was worked out as an experiment
in the course of reviewing the present series. The intention was to separate different
liistological pictures which might give rise to the different cell types found in
smears.

Several authors divide epithelial dysplasia of the cer\'ix uteri into two cate-
gories, basal cell hyperplasia and carcinoma in situ. In basal cell hyperplasia, a
variable proportion of the -width of the epitlielium consists of basal cells and the
severity of the lesion is defined in terms of this proportion (Nesbitt, 1955). We
have seen very few examples of tliis straightforward prohferation. Those found are
colleoted as grade 1.

The verbal criteria for diagnosis of “carcinoma in situ'’ are much better defined
than the lesions themselves, and it is frequently reiterated that loss of normal
stratification is a constant and essential change (Galvin and Te Linde, 1949).
In our opinion, much depends on the -word “ normal ”, Bowen s disease of the s 'in
has a range from complete disorganisation of the epidermal pattern to the presence
of abnormal cells scattered in aU layers in an othenvise fairly well-organi^sed
epithelium. Equally, in the cell nests of an invasive carcinoma there is olten
stratification from cells of basal ty’pe at the margin to keratimsed squames at tlie
centre. The present classification indicates inter aha the degree to which stratifica-
tion is maintained and tliis, like the extent of cellular pleomorphism and mitotic
activity, appears to vary in a very gradual manner with no sudden steps from
grade to th?next. This is also uddely recognised (Foulds, 1958). It ^
custom to describe and illustrate one’s criteria with great care, the illustrations

DKVKLOl’MKNT 01' CAHCIXO.MA 01' TIIK CERVIX

loo

covering a single inicrosco])ic licld, wln'lc in ])racticc a diagnosis is readied b}’-
examination of the wliole of tlic material available. As regards illustration, the
same eriticisni is inevitable in this series, and it is recognised that the changes seen
may varx' widely in the same sjiecimen. In general, Imt not necessarily in detail,
the changes in the non-invasivc lesions are towards progressively greater “ ana-
plasia ” with increasing mmilier. and the number given in each case is tlie highest
applicable. We believe that, all workers in this field would regard onr grades (j and

7 as “ carcinoma i» situ that many would include grade o. and that a few would
also include grade 4.

The grouping of invasive carcinomata used hero has nothing to do with degree
of anaplasia as such, but with jircdominant. cell type and arrangement which it
was thought might give rise to variations in cell jiailern in cervical smear.s. Grade

8 includes tumours of llrodcr's Grade 1—1. provulcd that they conform to the
criteria mentioned. The tumours in grade 1 1 have a jiattern much closer to that
of “carcinoma in .fiht " than to tyiiical squamous carcinoma.

A-F.

B. Cijlnlogicnl Clo.vsipaitioit

The predominant abnormal cells seen in smears were, on review, given a
numerical claasification. This has been done only for the pnr])ose of the present
inve.stigation and, like the histological grading, is not intended as a new terminologj’.

InsufRcient evidence for suspicion of malignancy.

In all these grades cells were seen with nuclei showing hypercliromasia
and some or all of the other aberrations which are found in carcinoma
(namely enlargement, multiplicity, folding or wrinkling of nuclear
membrane, prominent nucleoli, aggregation of chromatin into numerous
evenly or irregularly distributed granules (chromocentres)).

Cells have profuse endoplasm and are pol,ygonal in outline (Fig. 12).

B. Rounded cells with regular cj-toplasin and distinct cell borders. Few
small forms with high nucleo-cytoplasmic ratio. (Bi- and multinuclea-
tion is common in this type) (Fig. 13).

C. As B, but until predominance of cells showing high nucleo-C 3 doplasmic
ratio and/or small irregularh'^ shaped forms (Fig. 14).

R. Xuclei verj’- variable in .shape and size, sometimes pj'’knotic. Cytoplasm
often highty keratinised. Bizarre .shapes (tadpoles, fibres) (Fig. 1.5).
Large nuclei, high nucleo-cjdoplasmic ratio. Cjdoplasm shows bttle or
no keratinisation, stains poorly, and is often indistinctly outhned, torn
or lost. Some aggregates maj^ be present (Fig. 16).

Auclei rather uniform and crowded, occurring mainlj’’ in aggregates.
Separate cells have torn cytoplasm, or are necrotic (Fig. 17).

The

i^orphol grades have been chosen udth the idea of making broad

w diffe tlistinctions not between isolated cells, but between cell-populatiorrs
graded ^^i the abnormal cells under consideration have been

the acc’r. "i. account has been taken of whether they are many or few, or of
C nu Mammatoiy cells.

I'shed svst™^^R grades are not devised to correspond with any previously pub-
variationo Previous authors have used similar categories although with

"S’ m nomenclature.

156

JI. M. BODDINGTON, B. H. COWBELL AND A. I. SPRIGGS

• X divides the cells desquamating from cervical carcinoma

mto U-o mam tj^ea. My, those of " early malignancy ” shon nnolear chTZ
but a more or less normal cji^oplasm (our grades A, B, G). These “ dyskanmtio ”
cells may be of superficial, navicular or intermediate, parabasal, or endocenical
type. _ Dyskarjmsis is shovm in cases of intra-epithelial carcinoma or in cases of
earl invasion, but the superficial and intermediate type may only reflect epitlielial
dysplasia, and are more likely to regress spontaneously. Secondly, the cells of
advanced nialignanc}'- ” show C5'toplasmic as well as nuclear changes, especially
elongation ; they also often occur in large clusters,

Graliam and her co-workers (Vincent Memorial Laboratoi^L 1950) have given
a rather different classification, into undiflferentiated cells ” whose cytoplasmic
borders are indistinct, and “ differentiated ” malignant cells ivith distinct c}i:o-
plasmic borders. The second group includes “ fibre cells ” and “tadpole cells ”
which Papanicolaou would classify as cells of advanced malignancy, and “ third
type differentiated cells ” witli a high nucleo-cytoplasmic ratio, which Papani-
colaou would include under “ dj^skarj’-osis ”, and come into our grade C. Graham’s
classification is followed b 3 >- Zinser (1957).

For Graham (1957) tlie distinction between third t 3 ’pe differentiated and non-
malignant d 5 ^skar 3 ''otic cells is one of actual measurement, the malignant cells
having a nucleus wliose maximum diameter is larger than its distance from the
cell border.

A 3 Te (1952) describes a “ precancer-cell-complex ” which corresponds roughly
to Papanicolaou’s “ dyskar 3 msis ” and our grades A, B and C, although some
“ d 3 ’’skar 3 mtic ” cells would appear to fall into Ayre’s group of well differentiated
cancer cells. Cases of intra-epithelial carcinoma either show “precancer cell
complex ” or “ cancer cells of pre-invasive t 3 ’pe ”.

It has been maintained (Nieburgs and Fund, 1950 ; Soapier, Da 5 ’- and Durfee,
1952 ; Reagan, Hamonic and Wentz, 1957) tliat a distinction between carcinoma
in sifu and invasive carcinoma can be made from the c 5 d>ological appearances.
Reagan and his collaborators have made numerous measurements of cervical cells
and report that invasion is associated vdtb a change from rounded to elongated or
caudate forms, and that this feature is particularly noticeable in the keratinising
grovdlis induced by carcinogens in the cervix of the mouse (Reagan and Went^
1959). The differences between the smear patterns of carcinoma in situ and
invasive cancer have been codified by Wied (1956), but the presence or absence 0
blood, pus and Doderlein’s bacilli are considered as well as purely c 3 fylogical
characters, and there must be few who feel any great confidence in making sue 1
a forecast. Mackenzie (1955) analysing the cytological appeara-nce in 27 cases 01

carcinoma w found in 15 a predominance of Graham s third type ^ .

ated ” cells, in 9 a picture of bizarre liiglily differentiated ceUs exactly as is seen m
advanced cancer, and in 3 marked “ dyskaryosis ” vdthout any ce s consi e

“^M^mack, Belovich and IMeger (1957) have classified

invasive carcinoma, and describe three main groups, the firs v 0 o v i --i-jy

pond to Martzloff’s (1923) terminology. The/; spinal-cell ” group covers t^ lugh^

keratinised pleomorphic types ; the transitional (the larges gr P)’

admixture of large and small forms ; and the small-cell group

most anaplastic loistological ty^pe. Carcinoma tn sifu could not be distin„

with any certainty from invasive cancer.

Ftrecntngoof opinions

DEVELOPMKXT OE CARCINOMA OF THE CERVIX

157

L llistolo^icnl grnilo 1

•1 cases
Topinions

Senses

Copinions

50i

50-

20 eases
SGopinions

21 eases
•llopinions

Senses

llopinions

50-^ 9

4 cases
Sopinions

50 -^

24 cases
48opinions

^ P®, C ‘ D ' E ' F
'-ytological grades

CVTO-HISTOLOGICAL CORRELATIOX

For every observation a histological
slide was graded as well as the smear taken
shortly before. If the two cjdological
observers gave different gradings, each was
entered ns a half. j\Iorc than one entrj’
by the same observer was similarly frac-
tionated. A se])arate histogram has been
made for each histological grade, showing
the distribution of cytological assessments
found in that grade.

The numbers in each cytological grade
arc e.xprcsscd as jicrccntages of the total
nnmbcr for that histogram, so that every
histogram totals 100. (The absolute
number involved is given on the right.)

Where several observations were made
on the same patient, they are treated
independently for this purpose.

Histological grade 0 has been omitted.
The cases were deliberately selected for
cytological abnormality, and obviously
the figures cannot be used to show the
distribution of C3dological findings in
cases with normal cervical histologJ^ It
is likelj' that these were biopsies in which
the relevant area was missed.

The following deductions can be made
from the histograms :

(1) Alo.st liistological grade.s have a corres-
ponding modal cytological grade, but
there is so much variation from this that
it can never be justifiable to deduce the
histologj' from smears. Even the most
sinister-looking cells can be shed from
areas of midlj' unstable epithelium.

(2) On the average, the histological range
from slight instability to anaplastic
carcinoma is matched by a C3’tological
range, but some tj^ies are more
characteristic than others. For in-
stance, cj'tological grades A and B
were characteristic of histological grades
1, 2 and 3, and were very seldom found
in cases of invasive carcinoma. On the
other hand histological grade 7, without
invasion, was associated with a very
similar distribution of cytological grades
to that shown bj' the most anaplastic
invasive carcinomata (Grade 11).
Moreover, well-differentiated squamous
carcinomata (Grade 8) were matched
on the whole bj^ lower cjdological

158

M. jM. BODDINGTON, E. H. COWBELL AETB A. I. SPRIGGS

fcilclf if anaplastic-looking examples of “ carcinoma in situ ”

It would bo a inty to give the impression that the ethnological appearances are of
no interest beyond indicating the need for a biopsy. They certainly reflect cellular
changes winch arc of biological importance. They do not, however, give anv clue
to the invasive properties of tlie deeper parts of the epithelium, of which' tlier
represent only the superfieia] laj^er.

OBSKRVATIONS ON CASES WITH EPITHELIAL INSTABILITY OF THE CERAOX

Si.xty-seven cases were seen in wliich tlie cervix siiowed an abnormality short
of invasive carcinoma, bnt classifiable in the histological grades 1-7 described
above. Because we do not know wliere to draAv the line between “ carcinoma
in siin ” and lesser abnormalities, the}^ will all be included under the heading of
“ epithelial instability ”.

In tliree cases an invasive carcinoma was found at least a 5 mar after the first
smear. One of these (Case 5) is included altliougli there was no histological evidence
of epithelial instabilit 3 ^ but grade E cells were present in smears taken two years
before the carcinoma was discovered.

There were no deaths or known recurrences follovring treatment.

In the majority of cases wliere smears -were taken after the hiopsjL and before
anj' otlier surgical procedure, the abnormal cells -were found to have disappeared.
This was observed in 15 cases, and tliese have continued to have negative smears.
This group represents either spontaneous regression or complete removal of the

EXPLANATION OF PLATES

Figs. 1-1 1. — Sections of corvicnl epithelium representative of the gradings used in this study.
(H. and E.)

Fig. 1. — Grade 1. xHO.

Fig. 2. — Grade 2. x 40.

Fig. 3,— Grade 3. xSO.

Fig. 4. — Grade 4. xHO.

Fig. 5. — Grade 3. xHO.

Fig. 6. — Grade 6. xllO.

Fig. 7. — Grade 7. xllO.

Fig. 8. — Grade 8. x 35.

Fig. 9. — Grade 9. XllO.

Fig. 10. — Grade 10. x55.

Fig. 11. — Grade 11. X55. .

Figs. 12-17. — Papanicolaou-stained cervical smears showing cells representative ot tae

gradinKS used in this study. X 350.

° Fig. 12, — Grade A.

Fig. 13. — Grade B.

Fig. 14. — Grade C.

Fig. 13. — Grade D.

Fig. 16.— Grade E.

Fig. 17. — Grade F.

Fig. 18. — Case 2, first smear. x350.

Fig. 19. — Case 2, smear taken three years later. x350.

Fig. 20. — Case 4, first smear. x350.

Fig. 21. — Case 4, smear taken a year later. x350.

Fig. 22. — Case 5, first smear. x350.

Fig. 23.— Case 5, smear taken two years later. xSoO.

Fig. 24. — ^Case 6, first smear. x350.

Fig. 25. — Case, 6, smear taken almost three years later. X 4oU.

Fig. 26.— Case 9, first smear. x350.

Fig. 27.— Case 9, smear taken six months after the first. X4i)U.

Pig 28. — Case 9, smear taken fifteen months after the first. X4oU.

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British .Iovrxal or Ca>ckr.

Vol. XIV, No. 2

Boddington, CowdeU and Spriggs.

V.,1. NIV. N’-.

BitiTisit JoUitsAi. or Can< i-I''

Boddington, Cowdell and Spriggs.

DEVELOrMENT OE CARCINOMA OE THE CERVIX

159

lesion at biopsy. In three other cases the smears were still jiositive after the first
biopsy, but became negative after subseqxicnt ones.

Usually the course of the disease rvas interrupted by .surgical treatment, and
nothing more can be learnt from these cases, Tlicre was, however, a grou]) of ten
cases in which, for various reasons, a persisting lesion was followed by repeated
smears for at least a year. In four of these the ]ieriod of observation was 2-.3
years, and in one was 5 years. These arc a selected group, but are of particular
interest because the literature contains very few details about the changes, if any,
to be found in the desquamated cells over jieriods of time. Is there a continuous
transformation towards more anaplastic types? Or are such transformations
sudden, punctuating long period.s of morphological stability? Or does the cell-
tj-pe remain unchanged, even if invasion supervenes? The following brief descrip-
tion accompanied by photographs show the different patterns of cellular behaviour
observed m this series.

A. Persistence of abnormal cells ivilhout cijlological jnogression*'

Case 1 (Reg. No. 35470) aged 33. — Complaint — irregular e.\ce.ssive bleeding.
Six smears were taken over a period of 31 years. The first four showed cytological
grades A -f C, but two taken in the last year .showed little abnormality. Three
biopsies all showed grade 6 lesions. Hjmterectomy was finally performed, and
sections of the cervix showed areas of grade 6 and of more extensive grades 3 and 1.

Case 2 (Reg. No. 34924) aged 49. — Complaint — irregular periods. The cervix
was eroded. The smear showed grade A cells (Fig. 18). She failed to attend for
biopsy and was next seen 3 years later. A further smear still showed grade A cells
(Fig. 19) and a biops}’- shoAved a grade 3 lesion. She is still being followed.

Case 3 (Reg. No. 29734) aged 38. — Complaint — irregular prolonged periods.
Normal looking cervix. Cytological grade C, A biopsy showed a grade 4 lesion,
one was not seen for 5 jmars, after which another smear showed abnormal cells
out now of grade B. A recent biopsy shows no abnormality.

Case 4 (Reg. No. 29653) aged 33. Complaint — vaginal discharge. A small
erosion was present. Smears graded Eh-F (Fig. 20), biopsy grade 6. The same
ce type was repeatedly found in smears taken over a period of a year (Fig. 21)
biopsy 10 months after the first was again grade 6. Hysterectomy
■"as hen performed, and no atypical epithelium remained in the specimen,
nla' ^0- 33994) aged 35. — ^First seen in early pregnancy with a com-

CTad* E discharge. There was a vascular erosion. Smears sho-\ved

later^' done at this time, and turn smears taken

simila^^ Pregnancy were reported as negative, but on review^ one of them shows
and a ^ years later similar abnormal cells were still present (Fig. 23),
^'■as then*p^^rf early carcinoma (grade 8). A Wertheim’s hysterectomy

was^emff n^ged 33. — Complaint of vaginal discharge. The cervix

followed shouted grade B-C cells (Fig. 24), biopsy grade 1. She was

sbe liad tx- ^ cervical smears for nearly three years, during whieh time

type of poll” pregnancies (one ending in abortion, the other ectopic). The same

u as always present, though fluctuating in numbers (Fig. 25). Thirty-

'Pt the lesions became^^'^r • alteration of the abnormal cell tj’pe to a higher grade, whether

or not 1

160

M. M. BODDINGTON, R. H. COWBELL ARB A. I. SPRIGGS

(gmdrsr’”' ctoinoma

Ca^ 7 (Reg. No. 32352) aged 37.— Complaint-dj^spareunia. Smears shoued
grade C cells and biopsy shoived a grade 1 cervical epithelium. During the follow-
g year two further smears were taken, without appreciable change, fnd two fur-
fclier biopsies shoired no atj^iical epithehum. Unfortunately no farther smSJs
were obtained during the follomng year, but a hysterectomy was performed two
years after the first smear on account of menorrhagia and pelvic pain and the
cervix contained a very small grade 8 carcinoma.

The series of smears is incomplete in this case, but the original cell type per-
sisted for at least a 3>-ear.

Case 8 (Reg. No. CG/3475) aged 29. — ^At post-natal examination a large cervical
erosion was found, and smears showed grade E— R cells. A biopsy showed a grade
7 lesion. As the smears continued positive for 2 months ndthout change of cell
cervix was amputated. The specimen could not be graded owing to
previous cauterisation. Subsequent smears still showed abnormal cells, but these
were of a different sort (grade B). Another biopsy 6 months after the amputation
showed no abnormality, but a third after a further 6 months showed a grade 5
lesion. The cellular picture remained unchanged, and a total hysterectomy was
performed, retaining the ovaries.

After this tlie smears from the vaginal vault have remained abnormal (grade
A-B). A biopsy taken from the vault 5 years after the first shows an unstable
vaginal epithelium (grade 5).

B. Progression of cell type during observation

Case 9 (Beg. No. 33443) aged 28. — Complaint of infertility. Normal cervix.
Smear showed cells of grade C (Fig. 26). Biopsy showed a grade 5 lesion. A series
of 15 smears were taken over a period of 15 months, during which a normal
pregnancy and deliverj’^ took place. Six months after the first smear the abnormal
cells began to become more crowded, with a reduction in their cytoplasm (Fig. 27).
The last smear of the series, taken 3 months post-partum, showed abundant grade
F cells (Fig. 28) and cervical amputation was performed. The sections showed a
grade 7 lesion.

C. Regression after persistence for a year

Case 10 (Reg. No. 29506) aged 27.— Complaint—djismenorrhoea. There was a
third degree tear, and she was admitted for repair. At this time a cemcal smear
showed grade C-E cells. Repeated smears showed the same tjqie of cells, and a
biopsy 8 months after the first smear showed a grade 5 lesion. The almormal cells
persisted for a further 4 months, when a second biopsy wty done. This sliowed
only chronic cervicitis, and all subsequent smears were nega,tive during an obsen a-
tion period of 4 years. Two further biopsies showed chronic cervicitis only.

This series of cases illustrates three points. n *.

1 The same cytological type often persists with only slight fluctuations
considerable periods of time, and does not show continuous progression touards

more anaplastic types. . - . , ,

2. Invasion can almost certainly supervene m areas of epitlieUai
although it is impossible to prove that the lesion u as not invasn e a

instability
the outset.

DEVEIOPMEST op OiRCIKOMA OP THE CEItvIX ""

TO , „f «,P above cases invasive carcinoma svns ovontually fomicl after at. least

E. and 7) and in none of H.en, was any eyiolofi.cal

’’“f'to oLfsTeSnotmal cells showed an nnmislaUMc cyiological ,, regression
The smeS w«e not all eomparahle and it is difficuft lo he sore n^lher l o

recorda definite change at U weeks pregnancy, hnt the ^

after deliverv^ showed entirely new appearances, with marked crowding ol nil i
and loss of distinct cytoplasmic borders. There was also a chai^ roi

grade 5 to grade 7 . This example suggests that mondiological all cratioiiR, w hen ^ y
Sour, proceed stepwdse rather than by a contimimis transformation. re
(19526):in a case studied wdth over 200 smears, found a ‘ slow progression ml.
the rather sudden disappearance of the abnormal cells. In Ins book (Ayrc 1
changes in cell tyT;>e are illustrated in “ Cell Behaviour fctudics cases 1 and .1,
but it is not clear whether the alterations were sudden or gradual ; case 0 showed
one distinct change, then long persistence of the same ty^pe. Jilany more casc.s are
required to settle this point.

DISCUSSION AND CONCLUSIONS

Because of the accessibility of the cervix uteri for smears and biopsies, more is
knowTi about the earliest stages leading to cancer at this site than at any other.
We are able to recognise lesions which, carry a measurcable risk (about 30-40
per cent) of sooner or later becoming malignant (Petersen, 19.55). As t)iis develop-
ment probably occurs in a random fashion, it does not seem to be possiiilo to
predict the course in any particular case.

Unfortunately, the picture has been confused by disputes about w'hethcr this
or that histological appearance should be called carcinoma or not. Except when
there is evidence of invasion, the question is one of ivords and not of facts. It is
unfortunate that the terms “ carcinoma in situ ”, “ intra-epithelial carcinoma ”
and “ stage 0 carcinoma ” have been introduced, as they are most difficult to
define and no two pathologists will agree about their exact use.

We recommend the term ‘ epithelial instability ’’ to cover all the histological
lesions illustrated in Pig. 1-7 . Basal-cell hyperplasia (our grade 1) might seem out
of place here, but since in two cases (cases 6 and 7) it preceded inr'asive carcinoma
by 2-3 years, w^e feel that it should be treated with the same respect as the other
grades which ive have illustrated.

In any case found to have epithelial instability of the cervix it is of the utmost
importance to discover whether there is in fact an early invasive carcinoma winch

authonties have suggested that a distinction can be
lade fairly reliably from cjdological evidence. We cannot confirm this. Our
cj tological gradings, while roughly reflecting the cell-types seen in the correspond-

SJS f,*;:? .ns n 'Vift ‘he pretence SSteof

.li ^ ^ g^^'^ses on the basis of cervical smears

Sudy^f ’ rinf bTo^v ^ carcinoma ; a full histological

casel^cre 2 should be made in every

of untreated oases ceased to have positive

“ te Heternrine

162

M. M. BODDINGTON, B. H. COWBELL AND A. I. SPRIGGS

hin probably there have

been examples of each. In ten cases a persistent lesion was followed for over a

Hnfnrr^ eventually found to have smaU invasive carcinomata

Unfortunately our figures cannot be used to assess the probability of development
of carcinoma ; selection was exercised at all stages, and the cases with persitent
lesions which remained untreated are only a smaU residue of the whole series •
also the folloiwup period is very short. Our experience lias however, been quite
consistent with the pattern shovm in Petersen’s (1955) series ; he found that in
approximately 50 per cent of cases ivith “ cervical precancerosis ” the lesion disap-
peared within a year, but that if it persisted, carcinoma developed within 8 vears
in 50 per cent (13 out of 26).

Are the lesions which regi-ess diiferent histologically or cytologieally from
tliose which persist? The analysis of our findings shows no morphological criterion
correlated with persistence or regression, nor with the subsequent development of
carcinoma. In practice the distinction between persistent and transient lesions
is easil}’" made by the use of repeated cervical smears. In the present series those
smears which became negative nearly always did so following the first biopsy ;
case 10 is the only striking exception.

The series of persistent lesions provides some interesting evidence for the
“ stepwise ” progression of preraalignant states. As the paired illustrations show,
the abnormal superficial cells shed &om areas of epithelial instability are not
steadily ciianging towards a more anaplastic appearance. In the relatively short
periods over whicli u'e have been able to observe them they have usually remained
true to tjqie, just as malignant cells generally do both in experimental conditions
and in human disease. In case 9 a sudden change was observed, consistent with
the development of a new cell clone. The discovery of invasive carcinoma in
two cases while under repeated observation was not associated with any altera-
tion in cell tjqoe. Evidently, it is unjustifiable to expect any cytological warning
before invasion supervenes.

The problem confronting the gjmaecologist in these cases is never twice the
same, and no generalisations can be made about the appropriate treatment of
“ epithelial instability ”. Above aU, no help can be expected from the study of the
abnormal smears or biopsies in deciding the prognosis of an individual case.
There is no urgency, and provided that the patient is kept under surveillance
tliere does not appear to be any danger. On the other hand, a lesion of this tjqje
Avhich does not disappear after biopsy is undoubtedly “ precancerous ”, in the
sense that cancer is sooner or later very likely to develop, and the cervix or the
uterus ought to be removed, and negative smears obtained, before the patient is
discharged from observation.

SUMMABY

Now that 10,000 cervical smears have been examined from gjmaecologiwil
outpatients in Oxford, cytological and histological material pertaining to carcino <
of the cervix and possible precancerous lesions has been reviewed. ^

The lesions found at biopsy have been given numerical gradings The differe
types of abnormal cells seen in the smears have also been , -^1

a?.d a comparison has been made between the cytological and
gradings. Although there is a rough correlation between certain cytologi

development op carcinoma op Tine cieuvix

]iy.\

histological tj^es, the presence or absence of invasion is not correlalcd v.-ith

of possibly prccanccrous lesions (including
carcinoma in situ ”). In 33 of these the cervix or uterus rvns removed surgu-aily
without any further obserr'ations. In 18 the lesion could no longer be detecK'd
after biopsy, having either regressed or been removed entirely by the hiops> .
Cenucal smears were persistently positive for a year or more in 10 eases, and
three of these eventually had invasive carcinoma.

Review of the series of smears from these persistently positive cases showed
that the abnormal cell t^yie remained remarkably constant tlirougliout , evmi oyer
a period of several years. There was one exception, where a sudden ]>rogression
occurred to a more anaplastic-looking type. In the cases where ii'ivasi\ c carcinoma
supervened, no alteration in cell tj^ie was observed.

The data from tliis series are consistent with the idea that epithelial instability
(basal cell hyperplasia, “ carcinoma fri siiu ” and related lesions) usually disajipears
without any treatment more extensive than biopsy, but sometimes becomes
irreversible, and in that case sooner or later develops without any cytological
warning into invasive carcinoma.

H the smears and biopsies do not become normal within the first year, the
lesion should be regarded as precancerous.

We should like to express our thanks to Professor J. Chassar Moir for giving ii.s
the facilities of the Nuffield Department of Obstetrics and Gjuiaccology during a
large part of this work ; and to hlr. J. Stallworthy for his constant suj)port and
interest. We are verj" grateful to Dr. A. H. T. Pvobb-Smitli for his advice and lielp,
and to the British Empire Cancer Campaign, from whom A. I. S. and l\l. It. B.
receive whole-time grants. We thank Mrs. J. Shimmin, Mrs. R. Hughes, Miss c!
Clarke and Miss G. Olah for their assistance.

Ayue, j E.-(1952a) ‘Can^cer Gv^ologj^ of the Uteru-s’. London (Churclull).-( 19526)
•Sdi. med. J., Nashville, 45, 915. i \

Babes, A.— (1928) Pr. med., 36, 451,

Carsox, R. P. axd Gall, E. A. — (1954) Amer. J. Path., 30 15.

Fomns, L. — (1958) J. chron. Dis., 8, 2, ’ '

GALrTsy G. A. axd TeLixde, Pv. W.— (1949) Amer. J. Obstet. Gynec 57 15

n , 354. ’ • Tl-exbull, L. A.~(1957) Amer. J. Obstet. Gynec.,

L, j., D. KKttoKK, J. S,_,195„

'ubkiotf' K HwSwT' ®, 629.

* ■ CCS. H. E. AXD Poxn, E. R.-(1950) J. Amer. med. Ass., 142, 221.

164

M. M. BOPDINGTON, E. H. COlVDELL AND A. I. SPRIGGS

Papanicolaou, G. N.— (1928) Proc. Zrd Pace Betterment Conf., 528.— (1954) ‘Atlas of
Exfoliative Cytology Cambridge, Slass. (Commonwealth Fund.)

Petersen, 0. — (1955) ‘ Precancerous Changes of the Cervical Epithelium Copenhagen
(Danish Science Press).

Reagan, J. W., Haaionic, M. J. and Wentz, W. B. — (1957) Lab. Invest., 6, 241.

IdevJ AND Wentz, W. B. — (1959) A.M.A. Arch. Path., 67, 287.

Scarier, J., Day, E. and Ddreee, G. R. — (1952) Cancer, 5, 315.

Vincent jMeaiorial Laboratory, Staff of — (1950) ‘The Cytologic Diagnosis of
Cancer Pliiladelphia (Saunders).

WiED, G. L. — (1956) Ainer. J. Obslet. Gynec., 71, 793.

Younge, P. a., Hertig, A. T. and Arbistrong, D. — (1949) Ibid., 58, 867.

ZiN.SER, H. K. — (1957) ‘ Die Zytodiagnostik in der Gynakologie ’. Jena (Fischer).

ADBNOGAUCINOMA OB THE UTERUS IN INFANCY

A. G. aiAETLNS

From the Alder Hey Children’s Hosfital, LiveripooH-
Received for publication March 17, 1960

It is difficult to obtain accurate information about adenocarcinoma of the
uterus in infancy ; references to it are rare and some reports are lacking m detail.

According to Speert (1947) only about thirty cases of cancer of the cervix
had been described in the first two decades of life. In the same paper that author
reported the seventh case of adenocarcinoma of the cervix in girls aged 12 or
under. He stated that no case with epidermal carcinoma of the cervix had been

recorded in that age group. , ,

Boyes, quoted by Hark (1958), reports what he beheves to be the filth case
of adenocarcinoma of the cervix in infants under one year of age •. an ll-months-
old child which presented -with a 3 weeks’ history of vaginal bleeding and was
found at laparotomy to have an inoperable growth. Treated with radiotherapy
(Cobalt Bomb) with some improvement, the patient died nfith diffuse metastases
1 1 months later.

Amesse (1932), quoted by Jolly (1955), described an adenocarcinoma “ in-
volving” the uterus in a girl aged 23 months. Lockhart (1935), quoted by
Speer (1947), described a papillary adenocarcinoma of the fundus in a 14-months-
old girl.

The fact that adenocarcinoma of the cervix seems more common than adeno-
carcinoma of the fundus in children is in keeping with the observation in adults
that the relative incidence of carcinoma of the fundus increases -with age. It is
well known that the cervix constitutes the major part (two-thirds) of the pre-
menarchal uterus and that in mature women that relation is reversed.

Speert (1947) postulates a theory to explain the non-occurrence of epidermal
cancer of the cervix in children. He states that the lack of oestrogen stimulation
in this age period maintains the cendcal epithelium in relative quiescence (the
tliickness of the epithelium is only a quarter or a fifth of that in mature women,
mito.sis are much less frequent and the functional zone between stratified squamous
epithelium and columnar epithelium is more stable). Although this is an interest-
ing hjqiothcsis it does not represent the answer and the solution of the problem
must he looked for amongst more general biological factors accounting for the
failure of epidermal tumours to develop in the young, not only in the cervix but
m the skin and other situations.

It. IS well known that most children’s tumours are embrj'-omas. The uterus is
no exception and so the commonest tumour to be found in infancy is the so-called
cm uymua sarcoma of the urogenital sinus ”, also knoivn as rhabdomyosarcoma
or sarcoma botrj’oides, winch originates most frequently at the upper end of the
v»g„m .„n,. l.lor i„™do the uterus. GrossllhSdfrefers toTShfci IriJh

* nddres : .\v. Antonio Augusto Aguiar, 114 R/C., Lisboa-l, Portugal.

166

A. G. MARTINS

siiigle^Simnom”.””"' Center bnt he has not seen a

^ a. e.

Case Report

liospitnl ta “W'atta m^treata^^

The day after her transfer to Alder Hey, operation was performed tlironah -

‘w ‘“‘er-/“ ‘-“o- o/theX^rl:

sectjons showed that the tumour wag nialienaut.

tlie bladde^^urpH ^^'^ separated at the symphysis, the ureters were divided and

tinuitv rp.v’ ™ removed in con-

tinuitj (rig. 3). A single enjarged Jjnmph giand was removed from the pelvis ; it

contained in its sinuses cells similar to those of tlie primary tumour. The ureters
were anastomosed to tlie recto-sigmoid junction.

I macroscopic examination the tumour was 4-5 cm. in diameter, firm, almost
hard and its cut surface was homogenously white, ivith no areas of haemorrhage
or necrosis. The tumour completely filled the uterine cavit 3 '' and extended down
into the cervical canal.

hL’croscopjf showed a fairly well differentiated mucus-secreting adenocarcinoma
with areas of greater anaplasia (Kg. 4), which had penetrated deeply through the
wall of the fundus and reached the peritoneal surface. The posterior bladder
Avail Avas not penetrated and no invasion of blood vessels ivas seen.

The child stood the operation Avell and apart from an intereurrent infection
Avith R. coli 055 (infantile gastro-enteritis) had an uneventful post-operative
recoverjr. Post-operatiAm radiotherapy^ Avas considered but not favoured. The
child Avas discharged 6 AA'eeks after operation. Eectal examination and Wood
chemistry performed immediately before discharge Avere normal. The patient
AAms passing urine per rectum about eAmry 2 hours and was fairljf continent.

She was re-admitted 1 month later because of diarrhoea and Ammiting, clinical
examination AAms negative and the symptoms promptly subsided. Because lier

EXPLANATION OF PLATE

Fig. L — Child before operation.

Fig. 2. — Tj^e of incision used.

Fig. 3. — Specimen removed at operation : bladder, urethra, vagina and cervix, ^rh/ch is
invaded by fcumoiu* tliroughout.

Fig. 4. — J\licrophotograph ; low power magnification showing the tj'pical appearance of the
adenocarcinoma.

ilartins.

ADElSTOCARCDsOJIA OF UTERUS IN INEANCY

167

blood urea was slightly raised an intravenous pyelogram was performed which
showed a normal left pyelogram hut no emdence of excretion on the
A fortnight later the symptoms recurred and a large mass was now felt in the
risht side of the abdomen ; rectal examination was still negative. _

Exploratory laparotomy was performed on June 1, when a large inoperable
tumour- mass was found filling the right side of the abdomen A large amount of
dark broum mucoid fluid was aspirated from the tumour and a biopsy performed.
The biopsy specimens were unforbmrately non-significant but the cytolopr m the
aspirated fluid suggested tumour tissue. The wound healed well but the child s
condition progressively deteriorated and, apart from general supportive measures,
no treatment was considered justifiable. The child died on June 29, 1959.
Unfortunately permission for autopsy was not obtained.

niscussiox

The tumour in our case extended to the peritoneal surface of the fundus and
involved the entire cervix. As often happens in advanced lesions, it is difficult or
impossible to ascribe the origin of the tumour to the body of the uterus or to the
endocervix and it is therefore preferable for purposes of classification to classify
our case as an “ adenocarcinoma of the corpus and endocervix ”.

The initial symptom (but unfortunately late in the disease) is usually vaginal
bleeding in an otheruise healthy child, in whom clinical examination is negative
except for the presence of a low pelvic, painless mass. The presentation of
embryonal sarcoma of the upper vagina may be similar and clitflcal distinction
impossible unless the characteristic colourless, grape-like, cystic masses can be
seen on vaginal examination (clinical or endoscopic).

Endoscopic examination can be performed even in small infants either with a
bronchoscope or vfth the McCarthy cystoscope, using a flow of saline through the
instrument to distend the vagina.

Vaginal or cervical ojdology can be a help in the diagnosis of malignancy and
its type but should not delay treatment.

Trcalmcnl

Considering that the very few cases reported have all died, it is difficult to say
how they .should be treated. By analogy uith adults, the folloumg remarks seem
justified. °

Teclmical ffifficulties due to size, etc., would preclude the use of intra-uteiine
radium, even m cancer limited to the cervix.

Radiotherapy in “ curative ” doses would be too harmful to pelvic growth if
given m this ap grpp, so that its very doubtful benefits in a relatively radio-
f t such as this one are certainly surpassed by the disadvantages
Radical lipterocolpectoray en bloc uith pelvic lymph node dissection wnnlrl
.ocm to bo tl,c moot adequate form of treatment Lmo“aI the

mnligm,,” "““*">7 unless they are inTolved in the

ai.ou.d';±ru~ <=wce, mt

teem h, „r,a,.sdorit.ed troll's:

16S

A. 6. IVIAHTINS

clinically involved in the tumour (Schakman
1J50), although a more conservative approach, namely simple total hysterocol-
pectomy, lias been used with success (Ulfelder and Quan, 1947 ; Gross 1956).
Metastases are relatively late but kill quickly once they have occurred.

This suggested difference in the extent of radical surgery makes it important
that a bopsy should be taken at laparotomy and the result of a frozen section
examination obtained. The pathologists may only be able to state that tlie
tumour is malignant (we have been unable to find any reference to benign tumours
of tlie uterus or vagina in children), but he may recognize the typical mesenchjanal
cells or rhabdomjmblasts of an embryoma or the mahgnant epithelial cells of an
adenocarcinoma.

Unfortunately, so far, results have been disappointing : no survival has as
yet been recorded for adenocarcinomas of the uterus in infancy, which are
extremelj'’ malignant and of much Avorse prognosis than in adults. So far onl\'
one case (Lockhart’s (1935) patient, aa'Iio aa^s treated bj'' radiotherapy and died
17 months later of renal failure due to invohmment of the bladder) has lived for
more than a year after the diagnosis Avas made.

Radical operation is performed through a combined abdomino-perineal
approacli, either Avith a vmrtical or a T-shaped skin incision (separation of the
pubic bones improAung the exposure).

STlMJIAEy

A further case of “ adenocarcinoma of the corpus and endocervix ” in a 12-
months-old child presenting AA'ith a 3 weeks’ history of vaginal bleeding is des-
cribed. The extreme^ rapid CA’^olution of this lesion in cliildren is emphasized as
Avell as its rarity.

The non-occurrence of epidermoid tumour of the uterus in children is noted.

The treatment advised is early and radical hysterocolpectomy e?i bloc vciih
pehdc Ijnnph node dissection through combined abdominal and perineal approach.

Radiotherapj’’ is not indicated, except for palliation.

I am indebted to H'Ess Isabella Forshall for alloAA'ing me to publish tin's case,
to Drs. EdAvard Hall and Jean Bouton for their adAuce in the patholog}- aspects,
to Jtlr. Rodney Green for the photographs, to Mr. Charles Fitzsimons for tlie
microphotographs and to hlrs. Isabelle Hunter for the typescript.

REFERENCES

Amesse, J. W. — (1932) Colorado Med,, 29, 31 1 . , ni,-] j i i,- mi n

Gross, R.— (195G) ‘ The Surgery of Infancy and Childhood , Philadelphia, ( .

Saunders & Co.)

Hark, B.— (1958) Pediat. Clhi. N . Amer., 5, 95. ^ .

Jolly, H.— (1955) ‘ Sexual Precocity ’, Springfield, Rl^Ch. C. Thomas).

Lockhart, H. A.— (1935) Amer. J. Obstet. Gynec., 30, ib.

Schakman, R. (1950) Brit. J. Stirg., 38, 26.

Speert. H.— (1947) Amer. J. Ofistet. Gynec., 54, 982.

Ulfelder, H. and Quan, S. H.-(1947) Surg. Ohn. N. Amer., 27, 1240.

169

THE AHSEHIC CONTENT OF BRONCHIAL ]\'HJCOSA
AND SUBIilUCOSA IN MAN.

A Comparison of Specrmens from Lung Cancer Victims and

Control Tissue

K. H. HOLLAND, A. R. ACE\H;D0, and D. A, CLARK

From the Medical Research Service of the Veterans Hospital, and the Surgery Department,
University of Texas Southwestern Medical School, Dallas, Texas

Received for publication March 3, 19C0

PoptjLations exposed to arsenic dusts and fumes have a high incidence of
lung cancer according to six recent reports by Liebegott (1949), Hess (1956), Lull
and Wallach (1956), Osbum (1957), Roth (1957) and Braun (1958). Additional
evidence by Satterlee (1956) and Holland et al. (1958) has shown a high arsenic
concentration in urban atmosphere, and a 200-600 per cent increase in the arsenic
content of most American cigarette tobaccos from 1932 to 1957. Thus the arsenic
inhaled from our environment, cigarette smoke, etc., becomes a potential carcino-
gen when deposited in our respiratory systems and is worthy of exhaustive
investigation.

There are no known analyses of the arsenic content of bronchial mucosa and
submucosa, however, Bailey (1957) and Sula and Zelenkova (1957) did determine
the arsenic content of parencbjunal lung tissue and bronchial Ijunph nodes. It is
interesting to note that the latter two investigators found 2~70-times more arsenic
in the bronchial lymph nodes than in any other organ in the body and the more
anthracotic the lung and bronchial lymph nodes the higher was the arsenic
content. Unfortunately, these studies did not include bronchial mucosa and
siibmucosa, where most, if not all primary lung cancers arise. Therefore, it was
felt tliat an arsenic analysis of these strata should be performed on autopsj^
specimens from lung cancer victims and from cadavers that showed no evidence of
neoplastic disease in the respiratorj’^ system.

iLVTERIAL FOR ANALYSES

'Ihc specimens in this study were removed from male cadavers whose ages
occnjiations and smoking histories can be found m Table I. The lower traefea’
carma mam stem and lobar bronchi were removed intact with their hmph nodes
from L, victims of lung cancer and from 23 cadavers who showed no emdence of
neoplastic disease m the respiratory system grossly or on histological examination
e specimens were removed within 10 hours after death and the mucosa with

Vtri SX V ?fT'* »"d PlacS ta a

.rentcl i,! a .i.nihr ‘

170

R. H. HOLLAm), A. B. ACEVEDO AND D. A. CLARK

TECHNIQUE FOB ANALYSES

About 1 g, of each wet specimen {which we found the ideal weight for analj-sis)
was placed on an ordinary' previously weighed tissue slide. The wet weight’^as
determined by subtracting the weight of the slide from the weight of wet tissue
and the slide. The specimen was then placed in an oxygen bomb and dried for
three to four hours at a temperature of from 60 to 80° C. while a continuous stream

Table I. — As^O^ Content of Bronchial Mucosa and Subimicosa

A. Specimens from Lung Cancer Victims

As.Oj p.p.m.
mucosa and

Case

Age

Occupation

Smoking history

submucosa

C. D—

Railroadman

2-3 pkg./day

5-9

J. A. D— .

Oil man

2-21 pkg./day

6-5

H. B—

Carpenter

2-3 pkg./day

6-4

E. K. S— .

Plumber

1 pkg./day

6-3

H. D—

Painter

U pkg./day

6-7

W. U-

Laborer

Unknown

2-3

E. J. J— .

Railroadman

1^2 pkg./day

13-7

L. H—

Presser

3 pkg./day

13-4

C. T. B— .

Paper hanger

2 pkg./day; pipe .

5-0

J. B. IV— .

Engineer

2 pkg./day

4-5

O.P—

Watchmaker

1-H pkg./day

1-3

B. W. G— .

JIail clerk

2 pkg./day

7-&

L. R. J— .

Club operator

1 pkg./day

6'5

J. N. 0— .

Photographer

1 pkg./day ; pipe ; .

5-6 cignzs/doy

17-9

\V. F. D— .

Telegraph inspector .

3-4 cigars/day; pipe .

3-0

Jlean

. . .

. .

7-14

L. D—

J. W. K— .

R. L. H— .

W. R— .

F. P—

W. W— .

T. 0. C— .

F. 0. M— .

S. R. Y— .

W. A. K— .

H. L. H— .

T. C—

B. B—

A. D-

G. A. B— .

A.H.E— .

J. B. C— .

H. M. N— .

E.E. T— .

E. E—

W. I. G— .

J. M—

J, R

Mean

63-6

B. Control Specimens

Carpenter

Fanner

Painter

Electrician

Cabinet maker

Sawmill worker

Janitor

Cook

Groceryman

Electrician

Clerk

Yard worker
Broker
Laborer
Farmer

Nightwatchman
Railroadman
Guard
Insulator
Cook
Carpenter
Service Station
attendant
Railroad agent

pkg./day
II pkg./day
1 pkg./day
Pipe and cigars
J pkg./day
Unknown
6-8 cigars/day ;

1 pkg./day
Did not smoke
8-10 cigars/day

1 pkg./day

2 pkg./day
i pkg./day
1 pkg./day
I pkg./day

1 pkg./day
Pipe

Unknown
Unknown
U pkg./day

2 pkg./week
1 pkg./day
Chewed; dipped

Did not smoke

1- 7

2 - 1
1-2

1- 3

0- 75

1 - 2

3- 0

4- 2

5- 9

4- 0

2- 7

3- 7

5- 7
JM

2-1

9-0

1- 4
7-7
7-1

2- 4

3- r>

4- 7

8-4

4-13

ARSEICIC CONTENT OP BRONCHIAL MUCOSA I'l

of oxj^gen flowed through the bomb and into an adsorption train. This step
assured our obtaining most of the rolatile arsenic that is general^ lost in drying
a tissue.

When the tissue was thoroughly dried it w^as removed from the bomb and the
drj^ weight determined just as the wet w'eight. The specimen was next removed
from the slide udth arsenic-free cotton and placed back in the oxygen bomb
between loose la 3 'ers of cotton. The remainder of the analysis was performed
exactly as described by Satterlee and Blodgett (1944).

Fig. 1. Gross speeimen and insert shon-ing the dissection plane for removing the nmcosa -with its

underlj-ing submucosa.

KebUUTS AXD DISCUSSION

nf Ztfif ^ results of our study. The occupations and ages at the time

of death trere comparable m the lung cancer and control groups and there were
no knoum industrial exposures to arsenic ® ^ nu onere uere

nttnlvcic ac* rf * • *1 I 1 • ^ ^ CtiniClxlfc XU our

ZfnoTaSSe "tS'T

Sallcrlco (1056), and Holland e( oi (1958) ham O'dllllart (1957),

lolwccos n-hicl, are used almost oijlLfveW in 'Z'priZ'' o'garette

oco.«,„„al,igI,,raa„i„„„„tent. Themein ia oZl

n.vc.atig.atom in 19,57 n-„s 15 /,g./g 7rTn m SZ '.o '=5’

»l owed in fooda. Spot chocfa 3 MnS. / ‘>™‘

lol.ora,„rv,„ ■ »5n showed values ranging frSts by out

172

R. H. HOLLAND, A. R. ACEVEDO AND D. A. CLARK

Kennaway and Urquhart (1952) and Carey, Blodgett and Satterlee
(1934) have reported a high arsenic content of the dusts in urban atmospheres.
Ihe former investigators found a higher arsenic content in industrialized communi-
ties than in residential areas and they also noticed more arsenic in the dusts
in the vdnter than in the summer months. These findings are further evidence
tliat carbonaceous combustion products are another important source of inhaled
arsenic and are another testimom'al to our supposition that arsenic is the most
important causal agent of lung cancer.

SUjMjMARY

1. The inhalation of arsenic dusts and fumes over a long period of time is noiv
thought to be an environmental cause of lung cancer. Six recent reports are cited
to confirm this observation.

2. The mean arsenic content of the bronchial mucosa and submucosa of 15
lung cancer victims was significantly higher than the mean value found in the
23 control tissues.

This investigation was supported by a Besearch Grant from the National
Cancer Institute of the U.S. Public Health Sendee.

REFERENCES

Bailey, E. J. — (1957) Brit. J. Cancer, 11, 54.

Idem, Kennaway, E. L. and Ukquhart, M. E. — (1957) Ibid., 11, 49.

Braun, W. — (1958) H/sc/i. med. IFschr., 83, 870.

Carey, F. P., Blodgett, G. and Satterlee, H. S.— (1934) Indmtr. Engng Chem. [AmL),

6 3*^7

Gouldbn, P„ Kennaway, E. L. and Urquhart, M. E.— (1952) Brit. J. Cancer, 6, 1.
Hess, H.— (1956) Arch. Bin. Chir., 283, 274.

Holland, R. H., Wilson, B. H., Acevedo, A., McCall, M. S., Clark, D. A. asd
Lanz, H. C.— (1958) Cancer, It; 1115.

Liebegott, G. — (1949) Dtsch. med. Wschr., Ih : ; S55. i r u riita

Lull, L. and Wallach, A.— (1950) Montana State Department. Unpublish Data.

Cited by Heuper (1956) Fnbl. Hlth. Monogr. 36, 1.

Neubauer, 0.- — (1947) Brit. J . Cancer, 1 ; 92.

OsBURN, H. S.— (1957) Cent. Afr. J. Med., 3, 215.

Both, F.— (1957) Germ. med. Monthly, 11, 172.

Satterlee, H. S.— (1956) Neio Engl. J. Med., 254 : 1149.

Idem and Blodgett, G.-(1944) /adustr (^«fl .) ^6, ^ J.

SuLA, J. AND Zelenkova, V. (C^ec/^oslo^.'fl^•^a) 2, 31 /. Ci } ■

(1957) Brit. J. Cancer 11, 54.

TTT17 Ttn*m7 ■RESPONvSE relationship BET\M5EN I.HIj NUMBl'Hl
™ “oSSc miOE CELLS AND THE INCIDENCE OF
BLOOD-BORNE METASTASES

R BASERGA, P. B. PUTOXG, S. TYLER and W. B. WARTMAN
From the Department of Pathology of

and the Division of Biological and Medical Research. 'I he Argotnic .\a(wnal Lafxrit ,/,

Lemont, Illinois, U.S.A.

Received for publication April 10, 1000

It is knotvn that the incidence of blood-borne tumour mctastascs may be
influenced by many factors and by several experimental procedures (Baserga and
Baum, 1955 ; Wood, 1958). One of these determining factors is tlie number of
embolic tumor cells circulating in the blood stream. Zeidman, JlcCutchcon and
Coman (1950) showed that the number of lung metastases in mice was roughly
proportional to the number of tumor cells injected intravenously. More recently,
the frequent finding of tumor cells in random samples of venous blood from
tumor-hearing patients (Engell, 1955 ; Sandbert cf ah, 1958 ; Pruitt, Hilberg and
Kaiser, 1958) has indicated that a relatively large number of tumour cells may
actualljr be present at any given time in the blood stream of these patients.
These observations have prompted us to expand the investigation of Zeidman and
co-workers to cover a wider range of the dose-response curve, with the objective of
establishing a quantitative relationship between the number of embolic tumour
cells and the incidence of metastases. Because of the quantitative conditions of
the present experiment we thought it rvorthw'hile to investigate at the same time
other factors that have been said to affect the incidence of blood-borne metastases,
sue)] as the sex of the animal (Poel, 1957), the simultaneous injection of killed
tumor cells (Donaldson and Alitchell, 1959) or the pre-treatment A\ith viable
tumor cells (Hackmann, 1938) as well as the response of the reticulo-endothelial
system to the presence of metastases (Eoulds, 1932 ; Druckrey el al., 1939 ;
AVartman, 1959). For these purposes, different doses of viable Ehrlich ascites
tumor cells were injected into the tail vein of mice of both sexes, tw'o groups being
used to study the effects of the simultaneous injection of killed tumor cells or
previous injection of rdable tumor ceils. The incidence and number of lung
metastases in each group wms determined by actual count, and the w'eights of the
ungs, spleen, liver and kidneys Avere used to e.stablish a quantitative index of the
i-csponse of the reticulo-endothelial system to the presence of tumor metastases.

AlATEKIALS AKD JIETHODS

sexes and 4-6 months old which had been bred in the
Willard T Km Pathology of Northwestern University Medical School by Dr.

3 and gi™„

in tbi! ascites tumor, a subline of which has been nronavated

Lab„„torj. fe 6 years by sveeUy mbraperitoneal iniecSons £3*"

174

E. BASEEGA, P. B. PUTONG, S. TYLEE A2CD ly. B. lyAETJLlY

earners. Suspensions of viable tumor cells were prepared as follows : the Peri-
toneal fluid M'as aspirated 7-10 days after inoculation and centrifuged at 3000
r.p.m. for 10 minutes, the supernatant discarded and the tumor cells resuspended
in sterile isotonic saline in the desired dilution. The tumour cells were counted
in a heniocytometer, 5 to 10 counts being used for each dilution. Due to the
difficulties involved in obtaining round numbers of tumor cells, suspensions that
were as near the desired dose level as possible were used. The number of viable
ceils in the suspensions as determined by Sclireck’s method (1936), ranged between
93 and 98 per cent.

Susiiensions of non-viable tumor cells were prepared as follows : 7-10-days-oId
Ehrlich ascites tumor was aspirated from the peritoneal cavity of healthy carriers,
placed in glass tubes, centrifuged at 3000 r.p.m. for 10 minutes and the super-
natant discarded. Ten per cent buflTered formalin was added to the packed tumor
cells in a ratio of 7 : I and the suspension was placed in a refrigerator at 4° C. for
12-18 hours. The fornialinized cells were then centrifuged and washed 4 times
with normal saline solution and finally resuspended in sterile isotonic saline
in the desired dilution, ^^iability counts showed 100 per cent non-viable cells.

Onlj^ female mice were used to establish the dose-response ciuve. The number
of tumor cells injected and the number of animals in each group are shown in
Table I. For the second part of the experiment, on the incidence of metastases

Table I. — Incidence of Metastases in CAF^ Female Mice Injected hitravenoushj

with Ehrlich Ascites Tumor Cells

Number of tumor cells
injected ± S

None
£!05±170
14,3a0±700
93,200±G,S00
382,000 ±80, 000
o97,000±90,000
747,000±110,000
928, 000± 76,000
1,180.000±88,000
1,654, 000 ±140,000
],885,000±350,000
4, 526, 000± 180,000
6,7o0,000± 120,000
S,696,000±430,000

% of mice with

Number of

metastases

0-0

191
S J

-27 •

10-51 7,^ •
0-0/ .

16"^

25-0

33-3

52-3

80-8

100-0

114

100-0

815

100-0

—

100-0

—

100-0

—

100-0

—

= standard deviation.

= number of mice.

Number of
metastases per mouse
0-000

O-00Q/‘'

0-250

0-444

0-S64

2-192

6-062

14-250

40-750

>200

in mice previously treated with viable or non-viable tumor cells, only male mice
were used. The number of tumor cells injected and the number of animals m
each group are shoum in Table III. All injections, either of ma We or
cells were made into the tail vein, using a 2i-gauge
svuinae About half the injections resulted in local growths at the
in tlm tail or at the root of the tail. All animals that showed the f ^
of local growths were discarded, and were not included in the co p , ' j
Excent when othervdse stated, the mice were sacrificed by cervical (i^slocati
30 d4.. Sti X of tu4or cell*. Tl>e body .■e.ght and the ,ve,gh<s of

XUJIBER OP EMBOLIC TUMOR CELLS AND METASTASBS J

the lungs, liver, spleen and left kidney were deterinined for each animal. The
lungs were examined and the number of grossly visible metastases eounted by
two different observers. Precise counts were not possible when the number ot

metastases in both lungs was above 200. ,

The number of tumor cells in a given weight of packed Ehrlich ascites tumor
was determined as follows ; 5 ml. of tumor cell suspension, from 8-day-old
peritoneal growths were measured in a calibrated pipette and the number of cells
per ml. was determined as usual with a liemocytometer. After centrifuging and
discarding the supernatant, the packed tumor cells were weighed on an analytical
balance, the weight obtained being taken as the weight of the number of cells
contained in 5 ml. of tumor suspension. The procedure was repeated on 5 different
animals, and the results were averaged.

RESULTS

I. Dose-response. relationsMp)S

Table I shows the mcidence of lung metastases in CAPj female mice following
intravenous injection of Elirlich ascites tumor cells. Animals alive on the 30th
day of the experiment were saci'ificed. Other animals were autojjsied on the day
of death. AU animals of the groups receiving less than 1,180,000 cells were alive
on the 30th day, and the last 5 groups had mean survival times equal to 26, 20, 16,
15 and 14 days respectively. The incidence of metastases below 100 per cent
when plotted on probability paper, was linearly related to dose (Fig. 1). This
indicates that the distribution of susceptibilities to Ehrlich ascites tumor cells is
approximately normal with least square estimates of mean and standard deviation
equal to 512,000 and 394,000 cells respectively. The relationship between average
number of metastases per mouse and dose, for groups in rvhich a tumor count could
he made, is shown in Fig. 2 and is definitely nonlinear. Horvever, the difference
between the trend seen at small doses and that characterizing large dose groups
suggests that at least twm processes may influence the pattern seen in Fig. 2. It is
of interest to note that for doses equal to or less than 600,000 cells, the relation
jctween variables is essentially linear ; while for doses exceeding 600,000 cells
ue pattern of points accelerates even faster than a simple exponential function.

so for doses not exceeding 600,000 cells the group incidence predicted, based
on ioisson expectations wdth the observed number of metastases per mouse as
mean value, agrees closely wdth the observed incidence wdthin groups. On the
obse™*^d above 600,000 cells, the expectations greatly deviate from the

ascit'”T principal site of establishment and growth of Ehrlich

reflect injected intravenously, a wmight change in this organ should

and urn of the insult sustained by the organism as the dose is increased

dos-uL ' interpolation between the experimentally controlled

he a^noflnlf weight with injected dose (Table II) Avas found to

grouu.s indicator. Since the average Aveight of the lungs for

controls in frou 1 ^ seemed to vary about the mean oi the

bakon as tho I • of the lungs of the controls Avas

A linei Sint 1^3.2 mg

dose exists otr as per cent of the control value

s OA er a range of doses extending from 600,000 cells to approximately

176

baserga, p. b. putong, s.

TYLER AND W. B. WARTJLAN

to”*r or '“se te-

of the least square line indicates that the lung weight increases at a nnn=f !
rate of 0-165 grams per 1000 cells injected. The equation of the least

L = 3-55 + 0-165Z), 6 X lO^D 2 x 10 ^

Where L is the mean lung weight in per cent of the control
dose 111 thousands of injected cells.

( 1 )

value and D is the

VIABLE CELLS INJECTED (thousonds)

Fig. 1. — Probit transformation of tlie dose-incidence curve. Per cent of animals with lung
metastases, CAE, female mice injected intravenously with Ehrlich ascites tumor cells. Slope
= 0'002534 ± 0-00049S ; EDs„ = 512-9 ± 51-2 x 10= cells.

Although the dependence between lung iveight and mean number of metastases
per animal does not allow a simple explanation, an empiricall}^ derived functional
relationship between these variables is presented in Tig. 4. A log-log plot of
the variables shows a linear relationship between their log transforms. This
relationship at doses exceeding 382,000 cells is expressed b}^ the power law

M = 3-689(10-i«) (-)

where M is the mean number of metastases per animal and L is the mean huig
weight in per cent of the control weight. From equations (1) and (2), an empinca
expression of the dependence between number of metastases and dose can real i }
be determined.

1031*®®!!. OP EMBOLIC TUMOR

CELLS AKD METASTASES

177

VIABLE CELLS INJECTED (thousands)

Fig. 2. — Relationship bet'ween average number of lung metastases per mouse and number of
tumor cells injected. CAF^ female mice injected intravenously' with Ehrlich ascites tumor
cell.s.

Tai5LE II . — Mean Weights of Lungs, Sfhen,
Female Mice Injected Intravenously with

Liver and Left Kidney of CAF^
Ehrlich Ascites Tumor Cells

Body weight

, A.

Lungs weight Liver weight Spleen weight

Kidney

weight

injected

Xone

. 25

20-8

0-3 .

182

00.3

. 19

2o-9

0-5 ,

181

14,390

. 8

25-8

1-2 ,

161

9.3.200

. le

27-9

0-4 .

178

382,000

. 18

2G-0

0-3 .

154

.■)97,000

. 44

26-5

0-3 .

182

747,000

. 25

27-1

0-6 .

. 26

225

928,000

, IG

20-2

0-5 .

. 16

277

I,1S0,000

. 8

24-6

1-6 ,

. 8

344

1,0.74,000

. 20

2o-C

0-4

. 10

451

1,885,000

. 10

25-6

1-0

. 10

567

4„52C,000

. 14

24-7

0-8

. 14

572

0,7.50,000

. 11

25-1

0-8

. 11

665

8,090,000

. 6

25-6

0-9

. 6

687

.8uUq. injection

', 14

20-9

0-5

! 14

205

X Sx

1529 01

127

174

1301 29

132

165

1334 33

124

152

1472 43

159

173

1274 38

125

159

1539 80

158

181

1485 65

229 27 ,

191

—

224

12 ,

185

—

241

23 .

1656 41

327

12 ,

183

—

338 40 ,

1895 46

361

175

—

327

—

1925 145

504

189

>1 — number of mcasiu-e.s included in mean.

A = mean, in g. for body weight, in mg. for lungs, spleen, liver and kidney
St = standard error of the mean. muney,

Subq. injection : a group of mice with huge tumor growths at the root of the tail.

178

R. BASERGA, P. B. PUTONG, S. TYLER AlfD W. B.

WARTMAR

2. Response of the reticulo-endothelial system

_ Even though not a single metastasis was found either srosslv nr • n

m any organ except the lung, a significant resnnn^P or lustologically,

cells was noted in the spleen. The^weisht of the sni v.- tumor

number of tumor cells iniected CTable TT\^ T +i v tiighly correlates with

ob.o,„.e<, by variatoi'SS SfeSy leSrt'lSLt Z wt ‘S"?

sSen t)etween weight and dose is not present in the sample
Spleen weight and dose are linearly related (Fig. 3), and thus, by equation 1 ,'

DOSE IN NUMBER OF CELLS INJECTED (thousands)

Fig. 3. — Lung and spleen weights versus dose in CAFi female mice injected intravenously with
Ehrlich ascites tumor cells. AA^eight of control lungs : 173-2 mg.; slope = 0-1651 ± 0-0094,
intercept 3-55 ± 11-S8. AA^eight of control spleen: 133-6 mg.; slope = 0-0962 d: 0-0137,
intercept = 77-28 ± 17-17.

a straight line relationship betAveen the iveights of the spleen and lungs is implied.
In those mice in Avhich an improper intravenous injection resulted in a huge grovTh
at the root of the tail (Table II), the Ai'eight of the spleen tvas, on the average,
3 -7 times the weight of controls. This indicates that the spleen maj’’ respond to
the presence of tumor cells regardless of the site of tumor groivth.

3. The results in male mice

These are shoAim in Table III, from u'^hich it is apparent that the incidence
of metastases in male mice is considerably louder than in female mice, the 5 per
cent critical leAml being used as a measure of significance. It uill also ^
that AA'hen killed tumor cells are injected simultaneously AA-ith viable cells, le
incidence of lung metastases in male mice increases.

SrUlffiER OF E^roOLlC TUMOU CELLS ANO MI:TASTASF,S

175)

^^Hienraale nuce, are injcctotl. SO days a]iarl., wilh ( wo siinilnr (Iosoh of viiililo
tumor cells, the incidence of lung inctaRlascs is nhouf t wiee (I\a(. observed in iniee
injected nith a single dose. This seems to indicate that, jn-evions treatment,
with viable tumor cells docs not alter the response of tlu' Iiost, to a .Kceond
injection of enable tumor cells.

MEAN LUNG WEIGHT PER CENT

Fio. CONTROL WEIGHT

1 * ± Ov08, intorcopt, =

With the procedure' ^^rvivinff at the site of arrest

1 n,l

‘'^tleclifferenc- L Percentage dry weight of tL ^ average of .703,000 colls
tamor (18 per cent dry wlightr T, th7

;• As the mean weight of the lungs

180

BASEKGA, P. B. PUTONG,

S, TYLER AND W. B. WARTIAAN

of CAFj female mice was found to be 173-2 me- anv in

tins figure will give a rough approximation of tlie uLber of tumor cJlfpresStb
the lungs. Parallel studies using tritiated thymidine (Baserea ^

Halvorsen, mo) have sho>v„ tlfat EAT ce JinjeSd wZioS 'X

1 00 Der’oe'i5t*X'i “ "iooWing time of 20 hours, and uiti!

Z jtl M I? ® possible to calculate

froni these data_ the approximate number of tumor cells injected that actuallv
survived at the site of arrest and developed into a tumor metastasis This may be
expressed by the equation ■’

( 3 )

M'here is the number of surviving tumor cells at the time of injection, iV, is
the number of tumor cells calculated from the lung weight, and .r is the number of
doubling times between injection and death. Calculations have been performed
for the last 6 groups and the results are shoum in Table IV. These indicate that
the number of injected tumor cells surviving at tlie site of arrest is less than one
in one thousand.

DISCUSSION

The advantages of using ascites cell suspensions in the study of blood-borne
metastases have been pointed out in 1936 by Warren and Gates, and, more
I’ecently by Ambrus ef al. (1956). The advantages are niainl}’- tliree, i.e. most of
the tumor cells are viable, little or no stroma is injected irith the tumor cells, and it
is possible to reduce to a minimum the contamination with cellular debris which is
unavoidable vdth minced tumor tissue. These advantages are particularly
important in quantitative studies as showm in the present experiment (Table III),
in which the simultaneous injection of killed tumor cells increased the incidence of
lung metastases produced by the intraAmnous injection of viable tumor cells.

Although the technique of intravenous injection of tumor cells still remains
an artificial procedure vdien compared to the observation of spontaneous blood-
borne metastases (Baserga and Shubik, 1955), it should be noted that according
to Wallace (1956), metastases from intravenously injected cells can be obtained
only vdth those tumors that are also capable of spontaneous metastases. V itii
these qualifications, the following considerations may be made.

1. The dose-response relationship

When suspensions of EAT cells are injected intravenously in CAFi mice, in
doses ranging from as feiv as 905 cells to as many as 8-7 x 10® cells, the inci °
lung metastases increases as previously pointed out by Zeidman et al. ( o ),
Avith increasing doses. The relationship betAveen dose and mean number o
metastases, however, is linear only up to a dose of 600,000 cells, but or os
exceeding 600,000 cells the relationship deAuates from linearity and acceJen t
even faster than a simple exponential function. As the tumor cell suspensi ■
used in the present experiments Avere all prepared, by dilution, from ‘

pool, it must be assumed that the tumor cell population had ^ P

composition in the various doses. Then, the changing slope of le os - P ^
curve indicates that the establishment of a metastatic growth does not depena
solely on the presence of favored cells capable of surAuval at the site ot arre ,

181

OTMBER OF EMBOUC TUMOR CELBS AXD MEIASTASI.S
but that, at least wth doses exceeding 600,000 Apcl^'ihiroxp'lallr-

composition of the tumor cell popu a ion ■ jlh')-!) who. hv employing

?fa“SL differ from primer,, srorvlta l,y Imviog n Inplmr n..ml«T of

'“'ftt toliog to compare oor rcanlta triil. tl.o.=e oW„mc,l ly '''■"■I'''.';

.James (1959), who studied the dose-re.sponse relation.shpi of hAl cells injiUid
intraperitonealiy. They found too that the dose-re.s]TOn.se curve depar led from
exponentiaUty, and that the results could he best summarr/.cd by idoUmg the
distribution of sensitivities of mice against the dose, as we have done in r )g. .
Bj' comparing the two distributions of sensitivities, it would ajijiear that. ulu'U'as
850 cells are required when injected intraperitonealiy to produce tumor growths in
50 per cent of the animals, a mean number of .512,000 cells must, he injected intra-
venously to obtain the same percentage incidence of lung metastascs. As doses
increase, however, the differences seem to disappear, and approximately 1 .000,ti(i0
cells are requited to produce a 100 per cent incidence of either lung mctastascs
or peritoneal groudhs.

Fiom the present data, it may also be stated that, at doses exceeding .*182.000
cells, a linear relationship exists between the log transforms of lung weight in
percent of control weight and mean number of metastases, so that the increase
in lung weight may he used as an indicator of the mean number of mctaslnsc.s.
This is further confirmed bj" the linear relationship existing between miinhcr of
tumor cells injected and per cent increase of lung weight.

2. The resjponse of the reticido-endoihclial system

■Several authors, in the past, have suggested that the reticulo-endothelinl
sptem participates in the process of metastatic growth. Foulds (1932) found that
the incidence of metastases in the lungs, liver and spleen from Brown-Pearce
tumors mcreased considerably when the rabbits had been previously injected
vuh ti^Tan blue. Brouwer (1938) obtained similar results with a single injection

sLn in using higher doses of Thorotrast, Im coidd not

shou auj increase m susceptibility. The increase in the incidence of metastases

m 1® Vl\ J ‘"“‘“=‘“0" “f ‘he animal host (Cirio L SS

M m, J r. ‘'•34) has also been attributed to the denrS

l»h : TwS J O" metastases (Pomeroy,

ro,,s i,,jectio,,s;f\;SirI™ sul,cuta„;

of two different transplantable rat tumoS^^th? PfenL^Tobr^”^^*'^""-

the .Jensen sarcoma (Stern and WiUheim ’ 19351 caremoma and

indicate a respon.se of the reticuio-eJSSi=i ^ Present data definitely

to the presence of tumor cells in the animal hS fect"^th°T
'^-veen the spleen weights and the lung we^Ss

182

R. BASERGA, P. B. PUTONG, S. TYLER AND W. B.

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1 me, of pricked EAT = 503*000 colls.

, Nvinibor of colls in difforonco
Number of surviving colls „(genorations“r '

jtoi^ibek oe embolic tumor cells and metastases

183

of the sBleen may he used as an indicator of the amount of tumor present in the
hoL Whether the hyperplasia of the reticulo-endothehal system can be regarde
as favorable to the host or not, our data do not indicate.

The results obtained in male mice

We have already mentioned that the simultaneous injection of killed tunaor
cells increases the incidence of metastases and that the pre-treatment of mice
with viable tumor cells does not have any influence on tlm number of metastases
induced by a subsequent injection of viable tumor cells. Perhaps of more interest
is the striking difference in the incidence of metastases, after intravenous in-
jection of EAT cells, between male and female CAE ^ mice. A high incidence oi
metastases from mammary carcinomas has been reported in estrogen-treated
rats (Nelson, 1944). Poel (1957) has found that the incidence of lymph node meta-
stases from chemically induced skin tumors was higher in female than in male
mice. To bring out these differences, it is probably necessary to use relatively
small doses, as previously suggested by Gross (1942), who had found sex differ-
ences in the response to the subcutaneous or intraperitoneal inoculation of a
transplantable sarcoma in mice, only when using small doses. These results
should not be construed, however, as implicating a higher susceptibility of females
to metastases in general, as the reason for the difference may well reside in the
particular tumor. The results show, however, the advantages of intravenous
injections of relatively small doses of cells in the investigation of the various factors
that influence the incidence of blood-borne metastases (Wood, 1958).

4. The number of tumor cells surviving at the site of arrest

It has been known for a long time that the majority of tumor cell emboli fail
to sundve at the site of arrest (Goldmann, 1897 ; Iwasaki, 1915 ; Zeidman et al.,
1950), and recent experiments in this Laboratory using tritiated thymidine to
label injected tumor cells showed that the percentage of Ehrlich ascites tumor
cells that survive at the site of arrest is not above 8 per thousand (Baserga et al.,
I960). Calculations based on the present experiments (Table TV) indicate that
the 8 per thousand figure should be revised dowmward, and that, in all probability,
at least with EAT, each metastasis originates from a single tumor cell.

T he data used in these calculations are not all of the same accuracy. The
doubling time of EAT, 20 hours, and therefore the number of doubhng times in
each group, are luioun with considerable precision, and the number of tumor
cell per mg. of packed tumor cells can be considered reasonably accurate the
standard deviation of the count not exceeding 10 per cent. The least precise of
Ibc data is the actual number of tumor cells present in the lungs which is based
Oil ho difference from the control weight, that is, on the assSi^tiorthat the
amount of norma lung tissue remains constant. ActuaUy, normal lung tissue is
1 1 part replaced by tumor tissue especially when the number of mp+poto

resulting corrections in the number of tumor

184

R. BASERGA, P. B. PUTONG, S. TYLER ARB W. B. WARTMAN

i.e. that the number of surviving cells is in the order of one or less per tlioiisuKl
and that most of the metastatic growths originate from single cells.

SmUJIAKY

The dose-response relationship between the number of intravenous^ injected
tumor cells and the number of lung metastases was investigated in CAF, mice
using suspensions of viable Ehrlich ascites tumor cells. The correlation was
linear for doses up to 600,000 cells, but Avith liigher doses the relation between
variables increased even faster than a simple exponential function, thus suggesting
a two-fold mechanism in the establishment of tumor metastases. The increase
in lung weight, for doses exceeding 382,000 cells, was linearly correlated to the
number of injected cells, and its log transforms were linearly correlated to the
logarithms of the mean number of metastases. At equal dose levels, the incidence
of metastases was much higher in female than in male mice, and the incidence in
males was also increased by the simultaneous injection of killed tumor cells.
Previous treatment ndth viable tumor cells did not alter the response of the host to
the subsequent injection of a second dose of viable tumor cells. The weight of the
spleen was linearly correlated to the weight of the lungs, thus suggesting a
quantitative response of the reticulo-endothelial system to the presence of tumor
in the lung.

We wish to acknowledge our debt to Dr. Willard T. Hill for his generous help,
and to Aliss Annette Serpico, for secretarial help.

This work was performed in part with the aid of a grant (P-142) from the
Illinois Branch of the American Cancer Society, and in part under the auspices
of the U.S. Atomic Energy Commission.

REFERENCES

Ambrus, J. L., Ambbus, C. M., BraoN, J. W., Golubekg. M. E. and Harrissos,
J E.— (1956) Ann. iV.F. Acad. ScL, 63, 938.

Baseega, R. and Baum, J. — (1955) Cancer Res., 15, 52. ^

Idem, ICtsiELESKi, W. and Halvorsen, K.— (1960) Ibid., m press.

Idem AND Shubik, P. — (1955) Science, 121, 100.

Beouweb, P. — (1938) Beitr. klin. Chir., 168, 616.

CiBio, L. AND Balestra, G. — (1930) Pathologica, 22, 451.

DEncKSEY, H., Hampeei, H., Heekbe, H. aee Eaeei, E.-(1939) Z. Knb,]or,a..
451.

Engell, H. 0.— (1955) Acto c7uV. sca.Bd., Supp]. 201, 1.

Flaks, J. and Gbynkraut, 3 .-~[nU)Acfa cancrol.,Bp. \, /6.

Foulds, L.—{1932) Set. Rep. Cancer Res Fd. Lond_, 10, .1.

Goldmann, E. E.— (1897) Bei/r. Chir^^ 18, 595.

Gross, L.— (1942) Proc. Soc. exp. Biol. A -I •> ^9-
Hackmann, C.— (1938) A. Krebsforsch 48, 169.

IwASAKi. T.— (1915) J. Path. Bact., 20, 85
Kaziwaba, K. — (1954)

Nelson, W. 0.-{1944) YaleJ. Biol. Med., 17,21 ‘.

PoEL, W. E.— (1957) ./. nat. Cancer Inst., 19, Wl,i.

KIBIBER OF EMBOLIC TUMOR CELLS AIsTI METASTASES

185

Pomeroy, T. C. — (1954) Cancer Ree., 14, 201.

Pruitt, J. C., Hilberg, A. W. and Kaiser, E. F. — (1958) New Engl. J. Med., 259, 1161.
Rabotti, G-. — (1959) Nature, Land. 183, 1276.

vSandberg, a. a., Moore, G. E., Crossmtote, L. H. and Schubarg, J. R. — (1958)
Cancer, 11, 1180.

ScHREK, R. A. — (1936) Amer. J. Cancer, 28, 389.

Stern, K. and WrELHEiM, R. — (1935) Z. ges. exp. Med., 97, 354.

Wallace, A. C. — (1956) Brit. J. Cancer, 10, 724,

Warner, P. and Jaaies, A. T. — (1959) Ibid., 13, 288.

Warren, S. and Gates, 0. — (1936) Amer. J. Cancer, 27, 485.

Wartjian, W. B. — (1959) Brit. J. Cancer, 13, 389.

Wood, S., Jr. — (1958) Arch. Path., 66, 550.

Zeidaian, I., McCdtcheon, M. and Cojian, D. R.— (1950) Cancer Res., 10, 357.

186

FURTHER STUDIES RELATING TO Tin? Tivirpt Tn \ mT/~»ATn
RADIATION SDBnVAL CURVE ™AT™oR TEEaSnt nr
CBA MOUSE LEUKAEMIA BY TOOLE-BOdI SuMol

H. B. HE^TT 42^D C. W. IWLSON
Frojn {he Westminster School of Medicine, London,

Eeceived for publication February 9, 19B0

CBA mouse leukaemia used in the present experiments was described
previouslj'- (Hemtt, 1958). Evidence was then presented to show that the CBA
host mice to wluch the leukaemia was transplanted exliibited no detectable
immunological reactivity to the leukaemia cells. Tliis host-tumour system
therefore provides an ideal model for the examination of certain radiobiological
concepts pertinent to the radiotherapy of autologous tumours. The annals
of clinical radiotherapy are in themselves sufBcient evidence that no immuno-
logical resistance against autologous tumours in the human host can be relied
upon to destroj'' viable malignant cells that haA^e ■withstood the best endeavours
of the radiotherapist, and it cannot be too strongty emphasised that animal
host-tumour systems used to provide data relermnt to clinical radiotherapy should
be free from complicating immunological factors. Scott (1958) and IClein (1959)
have reviewed some of the complexities and fallacies that have often confused
the interpretation of radiobiological data obtained from experiments in which
immunologically reactwe hosts were used to detect vnable malignant cells that
have sundved irradiation.

Using the CBA leukaemia host-tumour system referred to, He-wdtt and Wilson
(1959a) determined a radiation survival curve for the leukaemia cells irradiated
in vivo. A linear relationsliip between dose of radiation and log survirml rate was
demonstrated up to the maximum dose of radiation used — 2000 r of ®®Co gamma
rays (corresponding to a surAmml rate of about 1/10®). The slope of the curve
indicated a mean lethal dose of radiation (Dq) of 165 r. Later evidence (Hewitt
and Wilson, 19596) showed that the leukaemia cells infiltrating the livers of
adv’^anced leukaemic mice breathing air had a radiosensitivity compatible ■with
their having been in a moderately well-ox 3 ^genated environment at the time of
irradiation ; when irradiated under anoxic conditions, the cells wmre shown to e
more radioresistant bj'- a factor of approximately 2-3. i i f

It -will be appreciated that the survival curvm data can be used to calculate
the minimum dose of radiation theoretically required to “ cure mice bearing
leukaemia cell populations of known size, provided that these cells have le
same radiosensitivity as the cells irradiated in the surviAml curim expenmen s.
The present experiments Avere designed to test directly our ability'’ to pre ic
“ cure ” rates in tliis ’w'^ay, and to explore such factors as might be e.xpec c
disqualify the simple application of the survival curve envisaged.

IBBADIATIOS OP MOUSE LEUKAEMIA

187

ulvterials akd methods

il/fce-GBA mice of either sex bred in this laboratory by sib-mating M-ere
used exclusively ; the mice Mere 2-4 months old when used for expenment.

Leukaemia.-— This vras a lymphocjdic type of leuhaenua ^hich arose spon-
tan«)u4v in a male mouse of the CBA colony Mhich provided all the mice used
n the experiments. The leukaemia was in its 74th to 108th senal passage Mhen
used in the present experiments. A detailed account of the characteristics of the
leukaemia, and of a transplantation hio-assay method for determming the mean
number of morphologically intact leukaemia cells required to com^y leukaemia
to half a group of injected mice (the TD50), tras pven previously (HcMitt 10o8).
An average TD.aO of 2 cells rras obtained in a series of 6 such assaj's of cells from

untreated leukaemic mice. ,

Irradiation.— Tox the -whole-body irradiation of groups of mice, these Avere
exposed in a “ Perspex box to s^Co gamma radiation delivered via a single
field in a single dose uniform to ± 5 per cent over a period of 16 hours. The
irradiated mice received 1-0-1-5 X 10® nucleated isologous bone marrow cells

intravenously -witlun 2 hours of the end of irradiation.

“ Badiation-killed cells ” consisted of fteshly-prepared single-cell suspensions
of leukaemia cells in .a per cent GBA mouse serum in Tyrode solution (density,
8 X 10® cells /ml.) which had been exposed immediately before use to a total
uniform dose of 6000 r ®®Go gamma radiation delivered over a period of 24 minutes.
The absence of viable leukaemia cells from the suspension was proved by the
injection of aliquots into higMy susceptible mice, aU of which failed to develop
leukaemia.

EXPERIMEXTS AXD EESTTETS

The effect of radiation-hilled cells on the growth of viable leukaemia cells

To measure sunnval ratios among cells irradiated in vivo, as was done for
determination of the surA'ival curve already referred to (Sewitt and Wilson,
I (Ibtki ). single-cell suspensions of leukaemia cells were prepared from the infiltrated
livers of leukaemic mice -within 30 minutes of their exposure to whole-body
irradiation, and these were injected intraperitoneally into groups of normal mice.
It is clear that adequate interpretation of the survival curve determined from
such data requires information concerning the possible influence of the radiation-
killed cells on the ability of the residual viable cells to convey leukaemia. Such an
mtluencc has been described preriously for other tumour-host systems (Revesz

In the present case, an inhibitory influence of the radiation-kiUed cells would
lead to overestimation of the proportion of leukaemia cells deprived of their
reprodnct.ye uitcgrity by the radiation. The existence of such an influence
nas sought m the follomng experiment: a counted single-cell susneS S
leulmenua colls from an nntreated leukaemic mouse waTassayed by^the fr trf
peritoneal uqcction of selected dilutions of the siisopnciAn ^ ^ r

norma! mice : the assay rvas performrifuticarLT™^^ '

immediately after injection of the viable cells each -moncA ““<^6 >'

a further intraperitmioal injection, ofo I ml of

..»»» .,r .1,0 rocdved 0-1 „f a Wens

188

H. B. HEWITT AOT) C. W. WILSON
months (a period t^vice as W f f f oWed for hvo

»it ’T£fi f

recorded The TMO valoe for™ ”rtee°

aren‘^rSdrSeirnr“2,^^^^^^

^-—^ssay of Viable Leukaemia Cells (a) Injected Alone ;
(b) Injected ivith Preponderence of Radiation-killed Cells

Jlenn dose of

Ratio of viable to

Incidence of leukaemia

viable colls

killed cells (fc)

(«)

lb)

125

1 ; 64 X 10*

6/6

5/6

12*5

1 ; 64 X 10*

5/6

1*25

I : 64 X 10*

0/5

3/5

0-125

I : 64 X 10*

0/6

TD50 :

5 cells

1 • 9 cells

two series Avere not significantly different from one another or from the average
figure (2'0) obtained for a previous series of assays in normal mice. It may be
added that no significant difference of survival times of the mice was observed
for corresponding groups of the two series.

It is concluded from this experiment that a preponderance of radiation-killed
cells, intimately mixed with the viable cells, does not influence the capacitj' of the
latter to reproduce the full disease in mice to which they are transplanted. Six
mice Avhich recewed l-G x 10® radiation-killed cells failed to develop leukaemia
after prolonged observation.

Assay of leukaemia cells in ivhole-body irradiated mice

When leukaemia cells are irradiated in vivo by whole-body irradiation of
leukaemic mice, and the treated mice are retained, it is conceiamble that, in addi-
tion to those cells xAdiich lose their reproductive integrity by a direct action of
radiation, others may do so later, as the result of indirect cytotoxic influences
resulting from relatively persistent constitutional changes induced in the host by
the whole-body irradiation. If such an indirect influence is active, the survival
ratios determined among the cells removed from the mouse verj' soon after irradia-
tion would fail to indicate the total damage to the mali^ant cell population which
would ensue among cells allowed to remain in the irradiated host. In these
circumstances, survival cimAms determined as preAuouslj'’ described (Hewitt ant
Wilson, 1959a), in which a sample of the irradiated cell population was removed
from the leukaemic mouse almost immediate^ after irradiation, would pmA it e
an unduly pessimistic estimate of the dosage requirements for successful ra 10
therapj’’. The following experiment was designed to reveal any inimical inmience.
capable of destroying viable leukaemia cells, which might be active m whole-body

irradiated mice. . , • r

A suspension of viable leukaemia cells was assayed m a control senes o ‘
mice and, in parallel, in an equivalent series of mice winch had received -0
whole-body ®®Co gamma radiation foUowed by intravenous isologous bone marrou
The assay was performed less than 6 hours after exposure of the irradiated mic

IRRADIATION OR MOUSE LEUKAEMIA

189

Table II. — Assay of Leukaemia Cells in N orrml Mice
and in Whole-body Irradiated Mice

Mean number viable
cells injected
1000
100
10
1

TD50 :

Incidence of leukaemia
»

In normal mice
5/6
5/6
2/4
1/4

5- 6 cells

In irradiated mice
4/4
5/5
3/5
1/5

4 ■ 6 cells

had been completed. The results of the two assays, given in Table II, showed
that there is no significant difference between the TD50 values obtained in the
normal and irradiated mice. Also there was no significant difference in the
survival times of mice in corresponding groups of the two series. It is clear that
no very persistent constitutional changes are present in the irradiated mice which
interfere ivith the reproductive capacity of cells which have survived the direct
lethal effects of radiation. These findings support the hypothesis that the survival
curve data can be used to predict dosage requirements for the successful radio-
therapy of mice harbouring malignant cell populations of known size.

Dose of whole-body radiation required to “ cure ” mice bearing known numbers of
leukaemia cells

Groups of 7 CBA male mice were injected intraperitoneaUy with dilutions of a
single-cell suspension of leukaemia cells prepared from an untreated leukaemic
mouse. The mean dose of morphologically intact leukaemia cells per mouse for
each group is shown in the first column of Table III. Within one hour of injec-

Table III, — Observed and Expected Incidences of Leukaemia in Groups of Mice
Bcariruj Known Mean Numbers of Leukaemia Cells at Time of Exposure to
IGOO r ’‘^Co Gamma Radiation

Mean number of

Mean number of cells
expected to survive

Expected

leukaemia

Ictiknemia cell.s

in mice after

incidence

injected

irradiation

(per cent)

1-1 X 10'

110

100

M X 10'

M X 10*

M X 10'

0-11

M X 10'

0-011

the potentially

leukaemic mice were

exposed

Observed
leukaemia
incidence
6/6 (100o/„)
6/7 (86%)
1/7 (14%)
0/7 (0%)
0/7 (0%)

t .. ..... UM uc.ivcieu in a single umlorm dose over a period of 16 hours
W ithm a fcB hours of the end of irradiation each mouse received intravenously
.approximate y 1 0 isologous nucleated marrow cells. The mice were then retained
for ohrorvn.io,. for . period of „t least 4 months, during whicTtoe ^ Sht
among II, c mico aarc investigated by post-mortem examination *

he™;“rs/rof:?eS:tsrr:SoS^^^^^^^^

.vnlespread inf., .ration of viscera mith lenkaemia cells! Sitg ^“ 3 !

190

H. B. HEWITT AWD C. W. WILSOxY

of whole-body radiatio 2 i had failed to eliminate the te+ai i .•
cells present in such mice at the start of irradiation Th leukaemia

deaths in each group is recorder! in the if i ^ uicidence of leukaemia

given in the VndVsS Zls of Me m wie”?

Fig. 1. — SunGval curv'e for CBA mouse leokaemia cells irradiated i>i vivo.

the h-radiated leulraemia cells retained their reproductive integrity after exposure
to that dose of radiation. The figures in the 2nd column of Table III are the mean
numbers of leukaemia cells per mouse expected to retain their reproductive
integrity after irradiation, calculated bj*- dividing the mean dose of cells injected
into the mice before irradiation (1st column) b 3 '^ 10,000. TJie expected per-
centage leukaemia incidence (3rd column) was obtained from a curve relating
mean dose of umTradiated leukaemia cells and the percentage of mice developing
leukaemia. This curve. Fig. 2, was constructed from data of quantitative trans-
plantation experiments reported previouslj’’ (Hewitt, 1958). Thus, the expected
incidences recorded in the 3rd column are those that would be obtained if the mean
numbers of reproductively intact cells remaining in the mice after e.xposure to
1600 r radiation were in accordance 'W'itli the survival curve. In spite of the sma

irradiation op 3IOUSE LEUKAEMIA

191

tt «pe»tS taddirot itlemTa aM in H.e

present radiotherapy experiment entered in accordance ndth

^ The points superimposed on the curve (Fig. 2) calculated

the ohsenmd eJ? nuSL {cd«mn 2 of Table III) for each irradiated

po^pTniioe* The departures ot the pointe trom the curve are weU uutiun the

Fio. 2. — Relationship between number of CBA leukaemia cells injected intraperitoneally and
proportion of injected mice developing leukaemia. The superimposed points relate to data
given in Table 111.

experimental error attributable to the assay method used. It is concluded that
the survival curve has accurately predicted the results of radiotherapy although
llic survival curve data used were obtained for leukaemia cells irradiated in a
quite different environment (the infiltrated livers of fully leukaemic mice).

DISCUSSIOX

Thc survival curve for CBA leukaemia cells irradiated in vivo (Hewitt and
]nr,0«) wa.s originally determined in the course of attempts to explain
our fiulure to cure leukaemic mice at an early stage of the transplanted disease
by then cxpo.sure to ««Co gamma whole-body radiation in doses up to 2400 r
followed by treatment with isologous bone marrow. This failure contrasted with

190

H. B. HEWITT AHH C. W. WILSON

folloM^s. From the survival^urve for thTlIT

(Hewitt and Wilson. lOSSarnSL SoiSpT,“?' '" ™

.600 r ..Co gamma radiation gave a snrtLi r:^o J’ t’JS CniJ: “.a5.“ «1

Fig. 1. — Survival curv'e for CBA mouse leukaemia cells irradiated in vivo.

the uTadiated leuliaemia cells retained their reproductive integrity after exposure
to that dose of radiation. The figures in the 2nd column of Table III are the mean
numbers of leukaemia cells per mouse expected to retain their reproductive
integrity after irradiation, calculated by dividing the mean dose of cells injected
into the mice before irradiation (1st column) by 10,000. The expected per-
centage leukaemia incidence (3rd column) was obtained from a curve relating
mean dose of unirradiated leukaemia cells and the percentage of mice developing
leukaemia. This curve. Fig. 2, was constructed from data of quantitative trans-
plantation experiments reported previously (Hevdtt, 1958). Thus, the expectc
incidences recorded in the 3rd column are those that would be obtained if the mean
numbers of reproductively intact cells remaining in the mice after exposure to
1600 r radiation were in accordance with the survival curve. In spite of the sma

IRRADIATIOj: OF MOUSE LEUKAEJUA

191

numbers of mice used, it is seen that there is a remarkably good correlation
between the expected incidence of leukaemia and the incidences obser\'ed in the
present radiotherapy experiment.

The points superimposed on the curve (Fig. 2) are entered in accordance with
the observed leukaemia incidences (column 4 of Table III) and the calculated
mean stuviving viable ceU numbers (column 2 of Table III) for each irradiated
group of mice. The departures of the points from the curve are well within the

the sunnval curve data used werf obtained for ]! f ^ of radiotherapy although
<|.nlc d.ffcrc„t c„vir„„„,e,.t (the irfiltrated Uvers of “ *

rp, . DISCUSSIOX

192

H. B. HEWITT AJJD C. W. WILSON

consideration of our survival curve in respect of the total leukaemia cell uou„ln
tion estimated to be present in our mice at the time they received experLLtil
adiotherapy revealed that the largest dose of radiation we used was not theoreti
cally sufficient to sterilise ’ a leukaemia cell population of that size. This
finding suggested that the survival curve might be used to predict quite accurateh-
tlie chance of a given dose of radiation being able to “ cure ” iffice bearing a'n
accurately measured number of leukaemia cells at the time of irradiation. We
appreciated, however, that such simple application of the survival curve data as
we envisaged could prove to be invalid on account of certain differences between
the conditions m the radiotherapy experiments and in the survival curve e.xperi-
ments. These differences ivil] be discussed further.

In the radiotherapj^ experiment the mice were exposed to whole-body irradia-
tion for 16 hours at a dose rate of 1-7 r/min. : in the survival curve experiments,
they were exposed to irradiation delivered in less than 30 minutes at 70-S0 r/min!
There is no reason to believe that such a dose-rate difference would result in
different survival ratios for the exposed leukaemia cells. On the other hand, it
is conceivable that in the longer exposure time some further division of the cells
might ensue during the earlier phases of exposure. The total number of leukaemia
cells to be “ killed ” would then be greater than the number of cells in a mouse
at the commencement of exposure. The possible error, however, is quite insig-
nificant, since interruption of mitosis would be expected to occur after the cells
had received the first few hundred roentgens of the total dose. Moreover, even
uninhibited growth of the cells during 16 hours would not be expected to result
in much more than a 2-foId increase in the number of cells (Hevitt and Wilson,
1959a).

In the survival curve experiments, the cells which had retained their reproduc-
tive integrit}' after irradiation were immediately removed from the irradiated
host and transplanted to normal hosts ; they were not, therefore, exposed to
possible inimical influences persisting in the constitution of the irradiated host
after irradiation. In the therapy experiments however, cells surviving the direct
action of radiation could have been exposed to indirect effects induced in tlie
treated hosts by the radiation. No exddence for such a persistent indirect
influence was demonstrated in an experiment specifically designed to reveal it.

In the survival curve experiments, the survWal ratios were determined among
cells infiltrating the livers of mice vdth advanced leukaemia at the time of irra la
tion. In the radiotherapy experiments the cells were in the peritoneal cavi } o
othervdse normal mice during irradiation. Since the radiosensitivity o le ce s
heavily infiltrating the liver was shovm to be compatible vdth then oemg m •
moderately well oxygenated environment during irradiation
19596) it is considered that cells dispersed in a small volume of fluid m tlie peri-
toneal cavity are in an environment equally well ox3"genated. The em iron *
oxygen tension, it may be mentioned, is the only extrinsic physio ogi < ‘

known to influence the radiosensitivity of the cells. In the
knowledge, therefore, there is no reason to suspect that the site i
be associated vdth a difference of radiosensitivity expressed m erms
lethal dose of radiation for the cells. , ,

Thus, experimental enquiry failed to reveal any factor whic
use of the sundval curve to predict the results practice,

and simple application of the survtyal curve in this way showed that, p

IREADIATIOK OF MOUSE LEUKAEMIA

193

wediction was as accurate as the statistics of the experiments
flunears that the rationale of the prediction procedure described ould be apphc
SK any sttem to the mean lethal dote ot radiation for ‘he feotae

cells and tL size of the mahgnant cell population were known. It is, ^herefore,
pertinent to discuss the imphcations of the findings reported here for other

tumour-host systems, especially human cancer. , x j

Puck Morkovin, Marcus and Cieciura (1957) have demonstrated that a varietj

1 -Rrhen irradiated in

of malignant and non-malignant cell types of human origin

® 1 Ti. KIa 4-V*ai

environments may prove to be a species characteristic only insignificantly attected
by cell type or by transformation of a cell type to the mahgnant state, h urther-
more, when suitable adjustments are made to allow for RBE difierences between
the qualities of radiation used in the human and murine experiments, it is clear
that no significant difference can be detected between the mean lethal doses of
radiation obtained for the human cell tj'pes irradiated in vitro and for a strain of
murine leukaemia cells irradiated in vivo (Morkovin and Feldman, 1959, 1960 ;
Hewitt and Wilson, 1960). Gray and Hewitt (1959, unpublished) have recently
shomi that the mean lethal dose of radiation for the murine leukaemia cells
irradiated under well-oxygenated conditions in vitro is not sigmficantly different
from that preidously obtained for the same leukaemia cells irradiated in vivo
in air-breathing leukaemic mice.

Thus, whilst it is desirable to obtain mes,n lethal dose values for additional
mouse cell tjqies irradiated under in vivo and in vitro conditions, there are already
strong indications that it might be well worthwhile to consider tumour dose
requirements for the “sterilisation ” of a wide variety of tumours, both in man
and the mouse, in terms of a single mean lethal radiation dose (approximate!}'
1 30 r, 250 kv X-rays or its biological equivalent of other radiation).

Assuming a common mean lethal dose of radiation for most human tumour
cells, several further factors require close examination before this value can be
used to assess tumour lethal dose requirements : estimation of the total cell
population of the tumour and of the proportion that are “ effective ” in the sense
that they can potentially form the basis of a recurrence ; estimation of the
jiroportion of “ effective ” cells under anoxic conditions at the time of irradiation ;
and the possible effect of fractionation schedules on the theoretical tumour dose
requirement. In inew of the very varied “ radiocurability ” of tumours it appears
that the question of o.xj'gen tension in the cell eniironment may be of decisive
importance, and it is a merit of the proposed approach to the problem that the
iniiiortance of this question is emphasised. It may be added that some of the

evonf’'r5n 1 ^ treatment of tumours may, in the

•' r *1’^ changes in the local tissue oxygen tension brought bv the

rcac ions of the tiimour bed to radiation. The intrinsic growth rate of a tumour
vould he expected to have a verj' considerable influence on the gross response of a

tumour dose roquiTement sinceTurrtval ^ to affect the theoretical lethal

of varmtion of cell radiosensitivity vith mitSicthasf
these factors would require histoiocdcal examinaSm nV

194

H. B. HEWITT AND C. W. WILSON

and not merely to assessment of the behaviour tendencies of the tumour derived
from Its histobgical pattern in relation to past experience. We believe tint
study of existing well-documented cases of radiotherapy along the lines
indicated could usefully be undertaken with the object of seeing whether the
results can be adequately explained in terms of survival curve data usiiw tlie
mean lethal dose of radiation referred to. “

SUMJIARY

A survival curve for GBA mouse leukaemia cells irradiated in vivo in mice of
the substrain of origin was described previously (Hewitt and Wilson, lOoOe).
Further e>^eriments are here described which were undertaken with the object
of examining various factors wliich could affect proper interpretation of that
survival cure. A bioassaj'^ method was employed to show that : a heaiy pre-
ponderence of admixed radiation-ldlled leulraemia cells did not influence the
ability of small inocula of viable cells to give rise to leukaemia after transplanta-
tion ; preliminary whole-body irradiation did not influence the resistance of
host mice to subsequent small inocula of vdable leukaemia cells. Groups of mice
bearing knomi numbers of viable leukaemia cells were exposed to 1600 r wholc-
bodj'- radiation and treated with intravenous isologous bone marrow. The
proportion of mice in each group which were thereby “ cured ” of leukaemia
was strictly in accordance with prediction from the original survival curve data.
This last experiment is deemed to exemplif 3 ’- a generally applicable thesis for the
radiotherapj'- of malignant tumours in autologous or tridy isologous systems:
that the curative dose of radiation for malignant cell populations of knovvn size
irradiated in vivo can be prescribed from relevant survival curve data. The
further problems lOrelj’’ to be encountered in such applications of survival curve
data are discussed.

AVe are grateful to h'Irs. J. J. Gough, B.Sc., (British Empire Cancer Campaign
Research Phjmicist) and to hlr. N. H. Pierce (Chief Technician, Phj'sics Depart-
ment) for assistance ndth the irradiation procedures, and to Miss E. Blake for
technical assistance vdth the biological w'ork. We are indebted also to the
British Empire Cancer Campaign for a whole-time research grant to one of us
(H. B. H.) and for financial support for the laboratory of the other (C. t\.

REFERENCES

Babnes, D. W. H., Cost, M. J., Loutit, J. F. and Neal, F. E.— (1956) Bnl. wed. J.,
ii, 626.

Hewitt, H. B. — (195S) Bri(. J. Cancer, 12, 378. ,, nnem Bril

Idem AND Wilson, C. W.— (1959a) Ibid., 13, 69.— (19o96) Ibid., 13, 6/o.-(I960)

J. Radiol., 33, 198.

IClein, G. — (1959) CaMcer i?es., 19, 343. r o j- ? qo oq9 Ibid 33,

Moekown, D. and Feldman, A.-(]959) Bnt. J. Radiol, 32, 282.-(ig0O) 10 , a., ,

Peck, T^^T., Mobkoiun, D., jMahcus, P. I. and Cieciuea, S. J.— (I9o/) d - 6 xp -

Reed, L. J. and Muench, H. — (1938) Amer. J.Hyg., 27, 493.

Revesz, L.— (1958) J. nat. Cancer Insi., 20, 1157. ’ Vol. VI.

Scott, 0. C. A.— (1958) ‘ Advances m Biological and Medieal Pli}sics ,

York (Academic Press Inc.), p. 121.

195

nATJOTTgof'-pNESIS IN THE PITUITAHY DWAHE MOUSE.
CAHCINO^NESK IN TO 2-AMINOELUOEENHL

E. BIELSCHOWSKY ai>’d HIARIANNE BIELSCHOWSKY
V ™ iTio Uiinl) Adam Cancer Research Department of the Medical School and the New
zZlald BraLh of the British Empire Cancer Campaign, University of Otago, Dunedin,

Wi/5 7,p.alri.nd

Heccived. for publication April 23, 1960

The comiective tissue elements of pituitary dwarf mice have been found to
respond to a subcutaneous injection of 0-5 mg. of metbylcbolanthrene m the same
way as those of their normal sized litter mates.

The purpose of the experiment described in this paper was to compare the
susceptibility of these mice to a systemically acting carcinogenic agent, ^-amino-
fluorene (AF), which alfects not only the parenchymal cells of a variety of organs
such as liver and mammary glands, but induces also neoplastic changes in the
transitional epithelium of the urinary tract.

Bickis, Estwick and Campbell (1956) did not find any spontaneous tumours in
their colony of pituitary dwarf mice. Nevertheless it was felt necessary to obtain
some information on the incidence of spontaneous neoplasms in old dwarfs and
in the phenotj'pically normal heterozygotes of our stock, so that induced tumours
might be distinguished from spontaneous ones. Therefore untreated animals
carrjnng the dwarf gene were used as additional controls.

Some preliminary results of our investigation have been mentioned at the
Symposium on Functional Components of Carcinogenesis held at Rehovoth in
1959.

JIATEKIALS AHD METHODS

The origin of the mice and their maintenance have been described in a previous
paper (Bielschowsky and Bielschowskj’’, 1959).

Whenever possible the experimental dwarfs were matched with normal sized
litter mates of the same sex.

Painting of the interscapular region with a 4 per cent solution of AF in acetone
was started when the mice were 8-12 weeks old. Ninety applications were given;
a No. 4 brush was used for the dwarfs and a No. 6 for the normal sized animals!
I he total dose administered to the former was approximately 135 mg. and to the
latter approximately 270 mg.

T he animals were killed when a palpable tumour was present or when decline
ni hcaltli nnade it advisable, and the remainder at the 52nd week of the experiment
1 heir maximum age was 141 months. ^ enmeiu,.

1 he untreated animals were kept in a separate room under the same conditions

oh 'szsjSi r'“

of t hcMii were more than 1 9 months old when autopsied. ^ ^

196

P. BIELSCHOWSKY AND MARIANNE BIELSCHOWSKY

The post examinations of the d^varfs were carried out under a ina<>ni-

lying glass. The histological methods used were those previously described
(BielschoY'slcy^ and Bielschowsky, 1959).

RESULTS

The average weiglit of the dwarfs at the start of the experiment was 7 g. and
8'.j g. at the time wlien treatment with AF w'as stopped. The corresponding values
for the normal sized mice were 23 and 26 g. Since the amount of AF given to the
dwarfs was approximately 50 per cent of that given to their phenot 3 picallj' normal
litter mates the former received, per ^am body weight, more than the latter.
Of the 55 dwarfs treated vdth the carcinogen, 39 survived for at least 29 weeks,
the time when the first tumour, a cancer of the bladder, was found. All other
tumours observed in this group occurred in animals killed in the 39th-o2nd veek
of the experiment.

In the normal sized litter mates the earliest tumour, also a cancer of the bladder,
was found in the 34th w’eek. The average age of these mice at death was 4j weeks
less than that of the dwarfs.

Table I shows the neoplastic lesions found at autopsy in untreated controls
(Groups I and II) and Table II those seen in the mice treated with AF (Groups
ill and IV). A comparison of the tumour incidence in the four groups reveals
differences between the du'arfs (Groups I and III) and the phenotjpically normal
mice (Groups II and IV) in respect to both spontaneous and induced tumours.
For instance, in the 41 normal sized mice treated with AF (Group III) 32 hepatomas
were found, but only 13 liver tumours in the 39 dwarfs of group tV. There was
therefore a highly significant association of normal body size with hepatoma
formation (F < 0-001). In the dwarfs both sexes were equally affected. Ingroup
III however there was a prevalence of hepatomas in the females, 90 per cent of

Table I . — Neoplastic Lesions Found at Autopsy in Untreated Controls

Control animals

, 1

Group I Group II

Neoplastic lesions found in

Total number of animals
Age in months
Animals with tumours :
Liver
Lung

Duodenum
Breast
Pituitarj^ .

Ovary

Lymph glands

Normal sized
untreated

28 (d H, ? 14)
13-25

Dwarfs
untreated
37 (d )7, $ 20)
21i-25i

— A « .

which developed tumours of the liver, whereas the incidence in the males of f

^°Ksfp?Sar^^ occurred in 2 normal

37th week of the expriment. Two weeks later a male dwar gr p

to have a liver tumour with a diameter of 3 mm. Three o

CABOISOOEKESIS IS PITUlTiEY DWAEF MCE

TABES L'^OW, Sem m Mice Trealei »;ft AF

Experinieivtal animals

Group IV
KeopAastic lesions found in

Total number of animals
Duration of experiment (weeks)
Animals with tumours in —
Liver
Lung

Pyloric region .

Breast
Bladder .

Kidney
Ovary
Uterus
Gall bladder

Kormal sized
treated with
A.F.

. 41 (d 20, 0 21)
34-52

32 (<5 13, $ 19)
5
12
8
4
1
1
1

Dwarfs
treated with

A.F.

39 (d 11. 2 22)
29-52

13 (d 7. 9 6)

those livers of mice of groups III and W -which at post mortera appeared normal.
Histological investigation failed to reveal neoplastic changes m those of nornial
sized mice but in 2 of the dwarfs minute lesions were seen. They -were formed by
the same type of cell depicted iu Fig. I, a pliotomicrograph of a macroscopically
recognizable hepatoma. This indicates that more hepatomas might have developed
in the dwarfs treated with AF had these animals been observed for a longer period.
Histologically there were no essential differences betw’een the liver tumours
found in groups III and IV, but in the latter, occasionablly foci of regressing neo-
plastic lesions (Fig. 3) were seen in livers -which at the same time contained
progressing tumours (Fig. 1 and 2). Only once, in a dwarf, were deposits of hepa-
toma cells detected in the lungs (Fig. I).

Spontaneous liver tumours were not found in untreated heterozygotes, but
3 of the dwarfs of group II had one or more macroscopicaUy recognizable nodules
in the liver. Only in one instance did a neoplastic lesion resemble those seen in
the liver of the experimental animals (Fig. 5). In the 2 others the histological
picture was of a more benign nature. One old dwarf had a single yello-wisb coloured
nodule composed of pale plant-like cells (Fig. 6), the other had 5 distinct nodules
the cells of which differed only slightly, by the basophilia of the c3rtoplasm,
from the surrounding liver cells. In this connection it seems worth mentioning
tliat areas of necrosis were not infrequent in the livers of old untreated dwarfs,
but amyloid present in some livers of untreated heterozygotes was never seen.

Benign papillomas of the gall bladder (Fig. 7) occurred in 2 of the normal sized
mice treated with AF.

Xo breast tumours developed in the d-u^arfs of groups II or IV, but 2 spontane-
ous mammarj' cancers were seen in the untreated heterozygotes and 8 occurred
in the normal sized mice treated with AF. The spontaneous tumours were t-roical
alveolar carcinomas and all the latter contained acanthotic areas.

here was little difference between the dwarfs and their litter mates in the
I espouse of the transitional epithelium to AF. In 4 animals of each group macro
.'-co|ueall\ recognizable bladder tumours were seen. Three were classified as
malignant because the muscular layer of the bladder was deeply invaded
such cancer occurred in a dwarf Tn ndrUUrA., ueepiy im aclecl. One

m a an art. in addition, there was one carcinoma of the

198

F. BIELSCHOWSKY AND JIAKIANNE BIELSCHOWSKY

pelvis in an animal of group III. No spontaneous tumours of the urinarv tmot
Avere found in the untreated mice, but benign papillomas of the duodenum omirred
m a few mice of ^oups I and II. The incidence of these lesions was coSS
liighei m the animals treated with AF. In the heterozj'gotes of group III V
tumours of tlie intestinal tract, situated mainly on eithi side of theyion!;
V eie found Two of these tumours had invaded the muscular layer (Fiff s)’
On the whole tliey were considerably larger than the spontaneous ones and the

three benign papillomas found in the dwarfs treated mth

AF (Fig. 9 and 10).

Tumours of the lung were not found in any of the dwarfs of either group II
or I\ although tin's was the most common spontaneous tumour in the hetero-
zygotes, 40 per cent of whicJi had single pulmonary adenomas. Of the normal
sized mice treated with AF, 5 developed benign lung tumours. In 3 of these
am'mals they were multijilc but were of a smaller size than those observed in the
much older mice of group I.

All the ovarian tumours found, including the one seen in an animal of group
III, are considered to be spontaneous benign neoplasms. Tliree of them irere
granulosa cell tumours and the fourth appeared to arise from the cells of the ovarian
stroma.

Discnssiojs'

In the rat ablation of thjToid (Bielscliou^sky and Hall, 1953) or pitiiitan*
(O’Neal, Hoffmarr, Dodge and Griffin, 1958) prevents tlie development of hepatomas
induced by AF or related compound, but tlie liver of the pituitarjr dwarf mouse
is susceptible to the action of this aromatic amine. In spite of the absence oi
growth hormone and in spite of arr extremely low level of thjwoid function, the
liver cells of the dwarf react to the carcinogen qualitative!}' in a similar manner
as those of normal mice or intact rats. The 'development of hepatomas is slowed
dowm, but not inhibited. In conjunction noth these findmgs the occurrence of
spontaneous liver tumours in 2-year-old dwarfs is of considerable interest and
stresses the fact that in pituitary dwarf mice, at least, a multiple hormone defi-
ciency does not prevent neoplastic grovdih in the liver.

The failure of the female dwarfs to develop breast tumours is easOy explaiue
by the virtual absence of mammar}' gland tissue ; they do not even have nipp
Although gonadotroplis are present in the pituitary of the dwarf mouse t wre u
hardly any indication of oestrogen secretion by the ovaries. Uterus andvagH ''*
always show'ed the picture of sexual immaturity ; also in our material t lere was

Fig. 1.

n'.

Fig. 2.-
Fid. 3.-
Fid. 4.-
Fig. 5.-
Fig. 6.-
Fig. 7.-
XSO.
Fig. 8.-
and E
Fio. 0 —
Fig. J.O.-

EXPLANATION OF FLAXES

—Hepatoma formed by large cells with eosinophilic cytoplasm from a dwarf of group

H. andE. x80. j ir vcn

—Another hepatoma from the same dwarf as Fig. 1. H. ana iii. ^

— A regressing neoplastic lesion — ^same dwarf as Fig. 1
—Hepatoma cells in lung of a dwarf of group IV. H^tid E.

— Spontaneous hepatoma from a dwarf of group II. H. arm -b. X

—Benign nodule from dwarf of group II. H. and E. X SO. „ . ^

—Benign tumour of the gall bladder froco, a normal sized mouse o gr p

m fX

-Adenocarcinoma of small intestine from a normal sized mouse of group
■Papilloma of smaU intestine from a dwarf of group op.

-Papilloma of duodenum from another dwarf of group . .n. ana r-.

British Journal of Cancer.

Vol. XIV, Xo.

Hielschoirsky and Biel.schoivsky.

CAECINOGEiSrESIS IN PITUITAEY DWAEE MICE

199

never any sign of luteinization in the ovaries. Therefore the four hormones which
normally stimulate the mammary glands, oestrogen, progesterone, prolactin and
growth hormone, if present at all, were secreted in oidy extremely low quantities.
In these animals the onlj'’ evidence for gonadotrophic action was the size of the
follicles. Some were larger than those seen in the gonads of females hypophysecto-
mized when sexually immature, but they never reached the size and appearance
of mature follicles, hievertheless in 2 ovaries of old untreated dwarfs non-
functional neoplastic lesions were found. In this context it might be mentioned
that in the male dwarfs evidence for gonadotrophic stimulation was more obvious.
The testes of some dwarfs had descended into the scrotum and in such glands
sperm was produced. However, stimulated secondary sex organs were rarely
seen in a male dwarf.

In view of the occurrence of spontaneous benign papillomas of the small
intestine in both groups of untreated animals it seems doubtful that the similar
tumours observed in mice of groups III and IV were induced by AT. The higher
incidence of these neoplasms, the larger size and the invasiveness of some of them
suggests that the carcinogen accelerated their development. In the dwarfs this
process seems to progress more slowly than in the normal sized animals (P = 0-013).

The absence of lung tumours in the dwarfs is surprising because in the normal
sized animals these neoplasms were far from infrequent. Of 17 untreated hetero-
zygotes, older than 20 months, 9 had pulmonary adenomas. It is well established
that the genetic constitution determines the susceptibility of mice to the develop-
ment of spontaneous as well as induced adenomas of the lung (Heston, 1942),
whereas, as far as we are aware, there is no evidence for hormones playing an
important role in their pathogenesis. Still, in view of the close relationship of the
experimental mice of groups III and which were litter mates, and the imtreated
heterozygotes of group I which were their parents, it seems difficult to believe that
non-systemic genetic factors could account for the difi’erence in incidence.

Further experiments are under way to test whether or not it is possible to
obtain lung tumours in the pituitarj' dwarf mice of our stock with the aid of other
carcinogenic agents.

STIMJIAEY

Tlie susceptibility of pituitary dwarf mice to 2-aminofluorene was compared
with that of their normal sized litter mates.

In^ the dwarfs the transitional epithelium of the bladder was found to be as
sensitive as that of the phenotypically normal animals, but, in the former liver
cells and the epithelium of the small intestine reacted in a significantly slower
manner to the carcinogen.

No tumours of the breast or lungs were found in the dwarfs whether treated

or imtreated. In the closely related normal sized animals these organs exhibited
benign and malignant neoplasms. ^ oxmonea

REFERENCES

Ricms. I., Estwick, R. R. axd Ca-Upbell, J. S.— (1956) Cancer 9 763

Rikusciiov^ky, F. Bielschowsky, M.— (1959) PnV. J. Cancer 13 ' zQ '>

Idem .\M) H.vu., B . H.— (1953) Ibid., 7, 358 cancer, 16 , d02.

Hilton, W. E.-(in42) not. Cancer Inst., 3, 79

xr.*,.. jjl, .A., H. E., B. G. Cbthk. A. C.-(1958) mi.. U,

200

SERIAL IRRADIATION OF MOUSE TUMOURS •
som CHANGES IN HISTOLOGY ^ GYmOGY

A. E. G. PEARSON

From the Department of Experimental Pathology, Monnt Yernon Hospital,

Northwood, Middlesex

Received for publication April 23, 1960

The metJiod of serially irradiating mouse tumours by transplantation into
iresh Jiosts between each ii'radiation, enables histological changes to be observed
divorced from the direct and indirect local effects of irradiation on the tumour
bed. Snellman (1935), eniplojnng this method, observed an increase in colhgen
in serially irradiated Jensen sarcoma in rats and interpreted tliis as indicating an
increase in the degree of tumour differentiation. He also demonstrated a decrease
in radiosensitivity in this irradiated line. It can be postulated that the increase
in degree of differentiation may liave occurred bj’^ selection from a mixed popula-
tion originallj’' containing more anaplastic cells, thereb 3 ^ assuming them to possess
a greater radiosensitivit}', or bj’’ transformation to a differentiating condition.
The correlation betiveen radiosensitivit}^ and these observations is obscure.
Gluoksniann (194S) favoured the opinion that the radiocurable tumours vere
distinguished by their capacity for increased differentiation after radiation ; tliis
is at variance with the observations of Snellman.

No changes in radiosensitivit 3 '- at the lethal dose level were found by Pearson
(1959cr) in four seriall 3 '- irradiated mouse tumour lines. Tliis communication reports
some elianges in histolog 3 ^ and c^'tohg}’^ observed in these lines.

MATERIALS AND METHODS

The methods emplo 3 ^ed in establishing serially irradiated lines of sarcoma 37
and two homologous tumours in RIII strain mice, have been reported previoiisl.y
(Pearson, 1959a). One ii-radiated line of sarcoma 37 received tumour sub-lethal
doses of 3000 r at each stage (“ B ” line) and one received half-ietlial doses of
2000 r (“ D ” line). Tumour sub-lethal doses were administered at each stage to
the homologous spindle-celled sarcoma BPl {“ F ” hne) and mammar 3 ^ adeno-
carcinoma ]\D^212 {“ G ” line). Tumour material for histological study was
obtained from transplanted post-irradiation tumours from each irradiation stage
and also from untreated sub-line passages of the 12th stage of the sarcoma <

ft g >> lin©

The tumour material was treated udth Susa fixative for 2 to 3 hours and em-
bedded in paraffin wax by chloroform substitution from alcohol. Sections ncre
cut at 6 /( and stained in haematox 3 >^lin and eosin.

RESULTS

No marked histological differences were apparent
spindle-celled sarcoma BPl (Fig. 2 and 3) and ocarcinoma iU and t

respective irradiated lines “F” and “G”. Small regions m & line tunio.

CHANGES IN SERIALLY IRRADIATED MOUSE TUMOURS

201

after three serial irradiations exhibited a decreased basophilia and a reduced
tendency to form acini, these areas were absent in control Ml 21- tumours (Fig.

The tendency to produce a liquid necrotic centre by sarcoma BPl timours
was greatly reduced in its irradiated line. Control tumours commenced to form a
necrotic centre on attainment of an area, measured externaUy, of about 60 sq^inm
This necrosis was usually delayed in “ F ” line tumours until an area of 90-100

sq. mm. was reached. i i ,

Histological variations in the cortical region were obsenmd between sarcoma
37 and “ B ” line tumours. An extensive cortical region, consisting of invasive
loosely connected tumour cells was present in controls (Fig. 6). B line turnours
at the 12th irradiation stage possesssed no comparable cortex, the edge of the
tumours consisted of packed cells and a thinner capsule (Fig. 7). Even in regions
where the connective tissue capsule had greater depth no invasion by tumour cells
occurred. These histological differences were ohsen’^ed until the 17 th irradiation
stage, when the series was discontinued ; were apparent at the 7th stage and u^ere
stiU present at the 32nd sub-hne passage (unthout further irradiation) of the 12th
stage. The 4th to 6th stages exhibited an intermediate condition.

No differences were observed between sarcoma 37 and its irradiated “ D ” line,
after 10 serial half-lethal dose irradiations.

Studies on the reticulin distribution in sarcoma 37 and B12 showed a sparse
loose network in the centres of both tumours, reported by Mackenzie (1958) to
be associated with anaplasia. The transition between this loose network and the
denser, more regular concentric patterns in the capsule was gradual in control
and abrupt in B12 tumours (Fig. 8 and 9).

Cytological comparisons between sarcoma 37 cells and those of its irradiated
“ B ” line revealed marked changes in nuclear morphology. Nuclei from control
tumour cells were usually kidney-shaped or irregular in outline ; the clrromatin,
stained uith Iiaematoxylin, comprising several small discrete bodies with a diffuse
staining of the nuclear sap (Fig. 10). Most cells of the B12 irradiation stage
possessed nuclei which had prominent usually single nucleoh, few chromatin
bodies and a less intensely stained nuclear sap (Fig. 11). These latter nuclei
conformed closely to the description by Caspersson and Santesson (1942) of “ B ”
cell nuclei, the control sarcoma 37 cells conformed to the “ A ” type of these
authors. This nomenclatiue is employed hereafter.

In order to determine whether the large staining bodies in “ B ” cell nuclei
were true nucleoli (plasmosomes) comprised of ribose nucleic acid (RNA) or false
nucleoli (chroniocentres) comprised of deoxyribonucleic acid (DNA), sections were
stained by the azan ” method of Heidenhain. These large chromatin bodies
stained red by this method and were therefore true nucleoh ; the hulk of
chromatin in “ A ” cell nuclei stained blue but usually 2 to 4 smaU nucleoli were
also present. Prominent chromocentres were not observed in “ B ” cell nuclei
I'hcse observations were confirmed by a negative reaction of the larae “ B ” cell
nucleoli to Feulgen stain. ®

Control tumours were found to contain some cells of the “B ” tvne aurl
irradiated line tmnours contained “A” type ceUs. Proportional cou^s were
o BlGrthe 06th fv " iri tumours from aU the irradiation stages (Bl

0 imfc "‘‘S" <>*‘2/26) ,„d from tL 10ft

uage oi tim irradiated D line (DlO). Five lugh power ( x 675) fields each were

202

A. E. G. PEAESON

PER CENT TOTAL TUMOUR CELLS

Fig. 1. — Proportions of “ A ” and “ B ” cells in sarcoma 37, serial irradiation stages of its
“ B ” line (B1-B16), 26th sub-line passage ivithout further irradiation from the l,Jth stage
(B 12-26) and the 10th irradiation stage of its “ D ” line receiving half-lethal discs at each
stage (DIO).

EXPLANATION OF PLATES

Fig. 2. — Sarcoma BPl. X 100.

Fig. 3. — Sarcoma BPl irradiated line “F ”, 7tli stage. xlOO.

Fig. 4. — Jlammari’- adenocarcinoma MV212. X 100. „ , , , • nf

Fig. 5.— Mammarj^ ndenocarcinoma MV212 irradiated line “ G ”, 10th stage, shoinng region oi

increased anaplasia. xlOO.

Fig. 6. — Sarcoma 37, cortical region. X 100. . , . , . mnsiilc

Fig. 7.— Sarcoma 37 irradiated line “ B ”, I2th stage, cortical region, shoinng reduced capsin

and loss of invasive properties. XlOO.

Fig. 8. — Sarcoma 37, cortical region, distribution of reticidin. xl20. -pulin.

Fig. 9.— Sarcoma 37 irradiated line “ B ”, I2th stage, cortical region, distribution of reticuim
X 120.

Fig. 10.— Sarcoma 37 showing typical “A” ceUs. x960. , yOSO.

Fig. II. — Sarcoma 37 irradiated line “ B ”, 12th stage, showmg tv-pical B cells, x-

British Jottrxai of Caucer.

Vol. XIV, No. 2.

Pearson.

203

CHANGES IN SEEIAHLY mRADIATED HOUSE TUHOUES

counted from cortical and central regions of tumours from each irradiation stage.
No consistent marked differences in proportionality were detected between the
two regions and the data rvere therefore combined, resulting in a total count ol
2000 to 2500 cells for each stage. Control data were obtained from tumour material
fixed at intervals covering the period taken to establish the irradiated line. No
significant differences were found in this control material.

The results of these counts are presented in Fig. 1. It can be seen that the
irradiation treatments favoured an early uniform increase in the proportion of
“ B ” cells up to the 11th stage. At this stage 25 per cent of the total cells were of
the “ B ” type, but after the next irradiation treatment this proportion had
increased to about 75 per cent and remained near this level at subsequent stages.
Sub-line passage (B 12/26) did not affect tliis proportion and it Avould appear that
a stable condition had been reached.

The proportion of “ B ” cells in the “ D ” irradiated line at the 10th stage
(Big. 1, DIO) was 7-3 per cent compared noth 3-8 per cent in control tumours.
The chi-square test of significance appbed to this data gave a probability of 4 per
cent ; the increase in “ B ” cells was therefore probably significant.

DISCUSSION

No marked changes in the degree of differentiation were observed after serial
irradiation — even in the less anaplastic tumours. A tendency in the irradiated
“ G ” line of kn^212 towards an increase in anaplasia is opposed to the observations
of Snellman (1935). Since no changes in radiosensitivity at the lethal level were
observed (Pearson, 1959a), in this instance radiocurability cannot be correlated
with the degree of differentiation.

The change in behaviour of sarcoma 37 “ B ” line cells in the cortical region
may have been linked vath the relative increase in “ B ” cells, although the same
histological change was present at the 7th stage when the proportion of “ B ”
cells had only risen to 8-3 per cent. It may be postulated that these cells were
unable to invade the adjacent tissue ; however tissue cultures from both tumour
lines showed both cells t 5 q)es in the form of actively migrating Spindle-shaped
cells. Twenty-five per cent of the cells at the 12th stage were of the “ A ” type
and it would be reasonable to expect some of these cells to show invasive properties,
as this condition was not obsers^ed, both cell tjqies must hai-e been altered by the
irradiations.

('aspersson and Santesson (1942) postulated that the “B” cell tjme was a
degenerate form of “ A ” cell ; this was based on evidence that “ B ” cells had a
lower protein content and occupied a more central position vithm the tumour
No zonal variations were found in the irradiated fine and if the “ B ” cells repre
.sented a degenerate form a reduced mitotic coefficient would be expected oi^e
o tho.r mcrea.scd proportion. Ifiitotic coefficients for (a) control and irradiated line
Utmonrs of (6) comparable age and (c) comparable size, the latter tumour Iffie

'iv k" S™"-th rate (Pearson, 19596), were 1-75, 1-74 and 1-66 respeo-

Usoh . 1 otal cel s counted were between 14,600 and 18,000 in each group anSe

in .he n.i.„Ho rn.o of ' A " SKte incase

for llio comjiarat.lc overall rate, ' ou d be necessary to account

204

A. E. G. PEAESON

Tlie glassy-cell type of mixed carcinoma of the cervix, described br
Glucksmann ^d Cherry (1966). appears to possess a nuclear norpholog,- oom«£

ablotothe E cells. Biese authors stated that this type of adenoacanlhmna

™s comparatively refractory to irradiation. Gusberg et al. (1954 and 1956) siitr-
gested that the proportions of “ A ” and “ B ” cells in cervical cancer could be
correlated lyith the clinical results from irradiation, a rise in the “ B ” cell pro-
portion indicating a favourable result ; the assumption ivas made that the “ B ”
cells^ Avere a degenerate form. The radiocurability of sarcoma 37 hmvever was not
significantlj' altered in its irradiated line, in spite of the considerable increase in
B cells. It wonld appear therefore that cells of tliis nuclear morphology may
not necessarily have comparable biological properties.

Measurements of DNA in control and irradiated line sarcoma 37 tumours
(Pearson and Atkin, 1960) showed that the tetraploid mode in control tumours
was replaced in the irradiated line by a mode corresponding to a triploid condition.
Allowing for errors in the selection of classes in the JDNA histograms and assuming
the morphologj’’ of hexaploid and triploid nuclei to be comparable, the proportions
of “ A ” and “ B ” cells in the two tumour h'nes are comparable to those of 4?!,
8??, and 3ji, 6ji cells estimated by DNA measurement. It w'ould appear that a
possible correlation exists betw'een ploidy and nuclear morphology in this case.

It is of interest that in the sarcoma 37 experiments the serial application of
irradiation produced permanent observable changes, not dependent on the con-
tinuation of the stress, which did not affect the radiosensitivity of the tumour.
This suggests that a selection or change by irradiation of certain ceU properties
can occur without affecting the fundamental radiosensitive features directly
concerned with cell reproducibility or viability.

sinvrMABY

1. Histological and cytological comparisons have been made on parent and
serially'’ irradiated lines of sarcoma 37 and twm homologous tumours in BIU

strain inbred mice. . .

2. The tw'O homologous tumours showed no marked changes after serial

irradiation. . -inr-u-:’

3. Regions of increased anaplasia were apparent in the adenocarcinoma iu\ - -

after serial irradiations. mnur-

4. Histological changes were apparent in the cortex of sarcoma u "

subjected to sub-lethal doses at each irradiation stage. ^ • u i rjrKT i

5. In this same line a proportional increase occurred in certain cells liav „

characteristic nuclear morphology. ^ i ivumont nf

6. Both these changes appeared to be progressive during the ®stabhstim
the irradiated line and w'ere not altered by sub-hne passage without lurr.

irradiation treatment.

7. The significance of these changes is discussed in relation to radiosensi .

ceU degeneration and invasive properties.

8. A correlation is suggested between nuclear morphology and pioiay .

The expenses of this research w'ere defrayed from a block grant by the Brif
Empire Cancer Campaign.

CHASTGES m SERIALLY IRRADIATED JIOUSE TUMOURS

205

REFERENCES

Caspbessoh, T. akd Santesson, L. — (1942) Ada radial., StochTi., Suppl. 46.
Get:cks3IAIvS', A. — (1948) Brit. J. Radiol., 21, 559.

Idem AKD Chebey, C. P. — (1956) Cancer, 9, 971.

CusBEEG, S. B., Tovell, H. jVL M., Esiebsok, R. akd Aluka, H. — (1954) Amer. J.
Obstet. Gynec., 68, 1464.

Idem, To'V’Ell, H. M. M., Lokg, M. akd Hill, J. C. — (1956) Ann. N.Y. Acad. Sci., 63,
1447.

Mackekzie, D. H. — (1958) Brit. J. Cancer, 12, 14.

Peaesok, a. E. G.— (1959a) Ibid., 13, 477.— (19596) Ibid., 13, 699.

Idem AKD Atkik, N. B. — (1960) Nature', Land., 186, 647.

SKELLJLiK, B. — (1935) Acta radial., StochJi., 16, 545.

206

THE NEOPLASTIC POTENTIALITIES OF MOUSE THYEOID
UNDER EXTREME STIMULATION

jM. S. ISRAEL AND I. ROSEJIARY ELLIS
From the Department of Pathology, Poyal College of Surgeons of England

Lincoln's Inn Fields, London, W.C .2

Rccived for publication April 20, 1960

H'i TERPLASiA of t]ie tJijToid gland aUJI follotr any procedure leading to tliiToicI
liormone deficienc}^ provided tJie anterior pituitarj^ is intact. This iias'been
demonstrated in rats and mice treated rvitli thiouracil (Astwood, Sullivan, Bissell
and Tj'slowitz, 1943 ; Gorbman, 1947) and also after the use of a lotv iodine diet
(Remington, 1937 ; Axelrad and LebJond, 1952). Attempts to produce thjToid
neojilasia b}' these means have been relative^ successful in rats (Hall and Biels-
chowsk}', 1949; Axelrad and Leblond, 1955), but the mouse responds much
less readily ; indeed serial transplantations of such thjToid tissue through several
generations of mice have usually been required to establish clear evidence of
malignancJ^

In the present experiment the neoplastic potentiahties of mouse thjToiil
gland under the stimulation of extreme induced thjToxin deficienc}’- has been
assessed by subjecting a group of mice to a combination of a thiouracil derivative
and a low iodine diet.

JlATERIAiS AND METHODS

One hundred 4-nionth'Old C57 mice, 50 males and 50 females, were segregated
by sex in metal cages, 6 to 7 per cage. The}' were then divided into 3 groups :

Group A — the control series, consisting of 12 males and 13 females,
was given a stock 41B diet and tap water.

Group B — consisting of 25 males and 25 females, was given the stock
diet and distilled drinking water with methylthiouracil added.

Group C — consisting of 13 males and 12 females, was fed on a special
low iodine diet in addition to the distilled water with methjdthionraci
added.

Methylthiouracil solution.— A stock 5 per cent solution was made by dissolving
10 g. methylthiouracil in 40 ml. of 2N-sodium hydroxide and diluting this to -00 mi-
noth distilled water. The use of sodium hydroxide was necessary because ol tne
poor solubility of nieth3dthiouracil in water. This stock solution was diliit
with distilled water to give a final concentration of 0-05 per cent met ij noun

This was given as drinking water to Groups B and C. ™nml

Lota iodine diet.— This was recommended by Dr. R. Pitt-Rivers p9o/, per <
communication), of the National Institute for Medical Researc i. consis

Hdiolemeal flour
Wheat gluten
Iron calcium flour
Drodisol yeast

1000 parts
660 „
200 „
140 „

NEOPLASTIC POTENTIALITIES OP MOUSE THYEOID

207

The iron calcium flour consisted of :

Calcium hydrogen phosphate (CaHPO.,) . . 40 parts

Hydrated ferrous sulphate (FeSO^.THjO) . - u

Wholemeal flour ..•••• ”

The mice were killed after 480 days and necropsies were performed The
th%woid glands were removed intact, together with the enveloped larynx and hrst
ring of the trachea, all of which was Aveighed together. The thyroids were not
dissected free of the trachea lest histological evidence of invasion might be lost.
The thjwoid glands and both lungs were fixed in 10 per cent forraol saline. Paraifin
sections AA'ere cut and stained AAuth Erhlich’s haematoxylin and eosin.

The pituitary glands were carefully removed for further studies, which will

be reported later.

RESULTS

Growp A. — The mice AA'ere in good condition and their Ai'^eights ranged from
20 to 32 g. The thyroid glands Avere small and uniform in consistency, and the
combined Aveights of thyroid and attached trachea ranged from 12 to 20 mg.

The histological appearance Avas one of regularly arranged large round acini
full of homogeneous, brightly eosinophilic colloid. The acini A’^aried in size but
they Avere all lined by a uniform Ioav cuboidal epithelium (Fig. 1). In none Avas
there any evidence of hyperplasia.

Group B. — The mice Avere in good condition and their Aveights ranged from
22 to 30 g. The thyi’oid glands AA^ere greatly enlarged and nodularity aars usually
obvious. Adhesion to neighbouring structures Avas not encountered. The com-
bined Aveights of thyroid and attached trachea ranged from 38 to 87 mg.

The histological appearance Avas one of profound hyperplasia of A^ariegated
pattern, though of fundamentally stereotyped structure. The basic feature aars
acinar hj’perplasia — groups of irregularly enlarged acini lined by an exuberant
loAA' columnar epithelium. Some acini AA^ere empty, others contained pale-staining
colloid and a fcAV Avere greatly distended Avith brightly eosinophihc colloid. The
hyperplasia aa’ss diffuse in extent, and in manj’’ areas there aars a pronounced
invagination of papillary epithelial processes into the larger acini (Fig. 2). This
change merged imperceptibly into the most characteristic feature of all, papillary
hyperiflasia (Fig. 3) Avhich Avas present to greater or lesser degree in all the treated
animals. It consisted of the inA^agination of copious strands of hj^perplastic
epithelium supported on thin cores of Avascular connective tissue into large cystic
spaces, most of AA-hich Avere empty, though a fcAV contained colloid (Fig. 4). The
cpilhelial ingroAvth aa’us so intricate that the acini AA'ere compressed into narroAV,
sinuous, eleft-like channels. Demarcation of papillarj^ areas into circumscribed
nodules AA-as common, and an appearance close to that of true papillary adenomata
was sometimes encountered in the most hjrperplastic glands (Fig. 5) The cells
hoAA ever. A\ erc uniform in appearance, though larger and paler than those of
noi I'lal acini. 1 he nuclear pattern was regular and mitotic figures Avere extremeh^

aie dig. (,). In the most liATcrplastic glands some of the Llls AA-ere verjr largt
and bad massiA’c nuclei (Fig. 7). ^

.A loss common variant Avas solid cell hAmernlasia in n-Lini, r

columnar colls with little tendenev to the fomation of central ?

m eireumscribod ncdules (Pig. 8). " ntral iumma appeared

208

jM. S. ISRAEL AND I. ROSEMARY ELLIS

In no case was there evidence either of infiltration into the trachea or tW
surTOiindjng muscles or of puimonary metastases.

conspicuous post-mortem findings except in 2 female
mice . one had a greatly enlarged spleen due to intense Ijnnphdd hyperplasia
and the other a large blood-filled simple ovarian cyst. The pituitary glands^m
not conspicuously enlarged in any of this group. ygianasvere

nf ‘Appeared to be slightly greater focal nodular papillaiy hjperplasia

of the thyroids m the females than in the males, but the difference was not out-

stfl II ciiilg.

6 rou 2 } C.~One mouse died naturally 4 days before the end of the experiment,
ihe remainder were in poor condition : their coats were dull and lustreless and
they were undernourished. This was reflected in their u'eights, which ranged
from 15 to 21 g. & > o

Great thjToid enlargement was again present, but the glands did not differ
markedly from those of Group B, with the exception of the animal that died
spontaneous!}’', in which the gland was replaced by a large cancer which had
infiltrated surrounding structures and caused death. The combined weight of
this mass and the trachea was 147 mg.; in the others the weights ranged from 40
to 90 mg.

The histological picture of profound papillary hyperplasia was augmented
by the presence of papillary adenomata in 1 1 of the 25 glands. These consisted
of circumscribed collections of proliferating cells arranged in a papillary pattern
(Fig. 9). Thej’ closely resembled exuberant papiUar}’ hyperplasia, but the crowded,
irregular arrangement of the cells and the distinctive cellular morphology indicated
focal neoplastic transformation. The cells ■r^’ere larger, more polygonal, their
copious cytoplasm was much more basophilic and their large, darkly-staining
nuclei were more irregular in shape and size than those of the neighbouring
hj’perplastic areas (Fig. 10). Ulitotic figures were scanty. Some adenomata were
very large and Avere composed of sheets of cells arranged in broad papfllae (Fig-
11). In these the possibility of early neoplastic change could not be excluded,
but there rr’as no erddence of local infiltration.

In the mouse Avith macroscopic cancer histological examination shorred

complete replacement of the thyroid by a poorly-differentiated adenocarcinoma

whose cells AA’ere arranged in large papillae in some areas and in irregular acini
in others. Colloid aa’us not found in these acini (Fig. 12). The lungs of this
contamed many metastases tlmoughout their substance and subpleurally , es
Avere rather better differentiated than the primarj’ tumour, for a feAv acini con-
tained colloid (Fig. 13). In the other animals no pulmonar}’ deposits were presen .

Pituitary enlargement AA’as conspicuous in this group, and m 8 mice ar
pituitary adenomata AA'ere present. One female mouse had a large anap as

carcinoma of the caecum. :

Of the 11 thyroid adenomata 8 occurred in females and 3 m males, ine

noma, hoAvever, occurred in a male mouse.

DisctrssroN

Both tMouraoiJ administration and a low iodine diet act by ”’*)!

thyfoxin synthesis. A deficiency of oircuiating tbjnoid hormone causes hypet

KEOPLASTIC POTENTIALITIES OF JIOUSE THYBOID

209

Tilasia of those cells of the pituitary responsible for the secretion of thjTotrophic
hormone (Russfield, 1955). Frank adenomata of these cells have hem
experimentally in mice given large doses of radioactive iodine (Burt Landmg a
Sommers, 1954). The thyrotrophic hormone induces hyperplasia of the thjTOid

^ It is of great interest to determine whether this hyperplasia can proceed to of
state of neoplasia, because such tumours would have been induced by an intrinsic
alteration of internal environment foUouing a derangement of hormone synthesis
rather than by the action of some extrinsic carcinogen. , „ .

In the rat such neoplasia has been produced quite easdj" (Bielschowsky, 19o5).
Indeed Hall and Bielschowsky (1949) demonstrated that, although 2'acetyl-
aminofluorene potentiated the neoplastic effect of thiouracil on rat thyroid during
the first year of administration, malignant tumours developed quite as readily
nithout the addition of the carcinogen after 18 months.

In the mouse it is much more difficult to produce thyroid tumours. It is
clear that an exuberant pattern of hyperplasia is characteristic, and even the
presence of discrete adenomata, valuable indications of progressive cellular
prohferation, will not suffice as incontrovertible evidence of neoplastic change.
Only definite infiltration of surrounding structures and the presence of distant
metastases can be accepted as positive proof of this.

Gorbman (1947) fed mice of 2 strains, A and C57, on a diet containing OT
per cent thiouracil. A vast series of changes were noted in the thyroids of these
animals, which were killed at periods varying from 7 to 566 days. Of the 22 sur-
vivors killed after 500 days, he foimd pulmonary deposits resembling thyroid
tissue in 7, all strain A mice. These little nodules were not accepted as true
metastases with neoplastic potentiality, but rather as fragments of hyperplastic
thyroid tissue that had acqxiired an intravascular position in a very active gland,
and had been then swept into the venous circulation.

Dalton, Morris and Dubnik (1948) fed 24 strain C3H mice on a diet containing
0-5 per cent thiouracil. After 362 days pulmonary deposits were found in 10
of them. Once again the intravascular position of these foci and their endothehal
investment was noted. Repeating the experiment with 32 strain C mice only one
instance of pulmonary involvement was encountered, though in these animals
nodular hy^perplasia of the thyroid was more conspicuous than in strain C3H mice
(Dalton, Jlorris, Striebich and Dubnik, 1950). It was essential to assess the neo-
plastic nature of this thyuoid tissue more conclusively, so Morris and Green (1951)
transplanted it into ymung mice on a diet containing thiouracil. After 3 to 6 months,
when the tissue had increased sufficiently in size, it was retransplanted into other
mice on a similar diet. After several transplantations there appeared lines of
tlp-roul tissue capable of growth in mice fed on a normal diet. IJltimatelv

some

now

of these tumours metastasised to the lungs and killed the animals. It could now
le concluded that autonomy has been attained, in that growth could occur without
any additional requirement of thyTotrophic hormone.

In the present e.xperiment a correlation between the degree of induced thvroxin
c icicncy and the gamut of hyqierplastic response has been made. The administra
“ ’-n’erplasia of a „LrtS„:

f,o,„

210

Sr. S. ISRAEL AND I. ROSEMARY ELLIS

fully established papillary hj^erplasia is folloived in turn bv o

"“t itoorrs "

riie diificult} in inducing malignant change in such glands is again emnhasi^Pd

Had fbe'" ”” I- ^ decisive cancerous ^transformatioJ’

Sivbf 1 ‘"’Sation been terminated later, it is quite possible that mali<^nancv
nf been encountered more frequently. To this purpose another” group

of mice is being treated similarly at present and in these the extreme thjaoxk
deficiency will be allowed to act for a greater period of time.

SUMMARY

One hundred C57 mice were divided into 3 groups :

(a) A control group of 25 mice fed on a stock diet and tap water.

(b) A gioiqi of 50 mice fed on a stock diet and distilled drinking water containing

0-05 ])cr cent methylthiouracil. °

After 4S0 daj's no significant changes were found in the thjToid glands of the
control group.

In the second group extreme h 3 q)erplasia of a t}q)icallj’^ papillar}^ tjpe vas
encountered, but there was no evidence of neoplasia.

In the third group focal papillarj’^ adenomata were found in 11 of the 25 mice,
but in none was there anj’^ tendencj'' to local infiltration or distant metastasis.
One mouse, however, did succumb to an adenocarcinoma of the th)Toid tliat
metastasised to the lungs.

The extreme difficultj’' in producing th 3 T 0 id neoplasms in mice even iinclcr
the most intense stimulation is demonstrated once again.

^Ve are grateful to Professor G. J. Cunningham for his interest and assistance :
to i\Iiss June Hunter, of the Medical School, Universit 3 ’- of Otago, Hew Zealand,
for help in planning the experiment ; to Dr. J. Craigie, of the Imperial Cancer

EXPLANATION OF PLATES

Fig. 1. — Normal thjToid glnnd in one of the control group. H. and E. xOo.

Fig. 2. — Pronounced acinar li 3 -perplasia with papillarj' invaginations into some acini, n.

and E. xC5. . , ..., 100:11

Fig. 3. — A later stage in the evolution of papillarj^ hj-perplasia from acinar lijTierpias .

H. and E. X65.

Fig. 4.— The pattern of fuUj'-developed papillaiylij-perplasia. H. and F. XOo.

Fig. 5. — A circumscribed nodule of papillary' hyperplasia resembling an a enom
cellular pattern is quite uniform. H. and E. X 65. j v ann

Fig. 6.— The regular cellular arrangement in papUlary hyperplasia. H. and E. x -ou.

Fig. 7. — Jlore pronounced hyperplastic pattern with some large cells havmg g
H. and E. x200.

Fig. 8. — Solid cell hyperplasia. H. and E. X 70. , i. ilw cells

Fig. 9.— a circumscribed papillary adenoma. The arrangement and morphology ot tl

is quite different from that of the surrounding tissue. H.andE. ^ lO.

Fig. 10. — The distinctive cellular structure of a papillary adenonm. il.^anu Ci.

Fig. 11 .—A particularly exuberant papiUap' adenoma. H. and E. oomc

Fig. 12. — ^A rather poorly differentiated adenocarcmiOTa shoumg a papillary p

areas and an acinar one elsewhere. H. and E. X '0- i-mttprn than tlic

Fig. 13. — A pulmonary' metastasis. It has a much better preserved acinar 1
primary tumour. H. and E. X 70.

British Journal or Cancer.

Vol. XIV, Xo. 2.

Israel and Ellis.

British Jouhxal or Cakckh.

Vol. XIV, Xo. 2.

Israel and EUis.

KEOPLASTIC POTENTIALITIES OF MOUSE THATIOID

211

Research Rund, for ad\ice about the low iodine diet ; and to Mr. A. L. E. Barron
for the photomicrographs.

REFERENCES

Asto'ood, E. B., SuLLI^^AN, J., BissELL, A. AND TvsLO^VITz, R. — (1943) Endocrinoloriy,
32, 210.

Axelead, a. a. and Lebeond, C. P. — (1952) Canad. med. Ass. J ., 67, 675. — (1955)
Cancer, 8, 339.

Bielschowsky, F. — (1955) Brit. J . Cancer, 9, 80.

Bust, A. S., Landing, B. H. and Sojcmers, S. C. — (1954) Cancer Bes., 14, 497.
Dalton, A. J., Moeeis, H. P. and Dcbnik, C. S. — (1948) -7. not. Cancer Inst., 9. 201 .
Idem, Moeeis, H. P., Steiebich, M. J. and Dubnik, C. S. — (1950) Ibid., 11, 391.
Goebjian, a. — (1947) Cancer Bes., 7, 746.

Hall, W. H. and Bielschowsky, F. — (1949) Brit. J . Cancer, 3, 534.

Moeeis, H. P. and Green, G. D. — (1951) Science, 114, 44.

Remington, R. E.— (1937) J . Nutr., 13, 223.

Russfield, a. B. — (1955) J . din. Endocrin., 15, 1393.

212

CARCINOGENESIS IN THE SKIN OF THE HEDGEHOG

F. N. GHADIALLY

From the Department of Palhohgrj, The University, Sheffield
Received for publication March 31, I960

It 1ms been sho^m (WJiiteley, 1957 ; Ghadially, 1958. 1959) that many
caTcniogen-mduced cutaneous tumours of laboratory rodents arise from pilo-
sebaceous follicles and are morphologically similar to the kerato-acanthomas
Ox man. 1 no-sebaceous follicles show considerable variations of morphology and
distribution in various animal species. The spines of the hedgehog are homologous
to the hairs of laboratoiy rodents and arise from A’^ery^ large follicles set yrell apart
from one another, while rodent skin is thickly populated by comparathmlj’ small
hair follicles. It Avas felt that the study of carcinogenesis in a sldn so different
from that commonly employed might yield A^aluable information regarding the
histogenesis of carcinogen-induced cutaneous tumours. Hitherto the hedgehog
has not been employed for the study'' of chemical carchiogenesis.

SrETHODS

The spines on the dorsum of six hedgehogs Avere removed with the aid of
nail clippers, and an area of sldn about 2 square inches was painted with a solution
of 2 per cent w/m' 9 : 10 dimethyl- 1 : 2 benzanthracene (DMBA) in equal parts of
lanoline and paraffin. A total of 30 applications of carcinogen was administered
OA'er a period of eleven months, and the animals were observed for a further
2-month period. Four animals died during the early stages of the experiment
{3 to 6 months). Of the tAvo that surAUA'ed one deA'eloped tumours during the
eleAmnth month ; the other died about a month later -without producing any
tumours. The animal with the tumours was killed approximately' 13 months
after commencement of the experiment. The tumours and pieces of adjacent
skin were remoA'ed and sections stained with haematoxylin and eosin AA’cre prepared
in the usual manner.

BESXJLTS

A number of tumours were produced on the skin of the hedgehog at the site
of painting (Fig. 1). One outgroAving papilloma (Fig. 2) was found
the epidermis. Typical type I kerato-acanthomas were not found but tlwe sn •
horns similar to that shoAvn in Fig. 3 were discovered. Since such a

placed horns with a cup-shaped base have been found to CA'oh'e
kerato-acanthomas in other species (Ghadially', 1958, 1959), Jt j

are here untnessing the same phenomenon. The base of one o J

seen in Fig. 1 was carcinomatous and had produced three small s ,

secondary growth in a lymph node. In two of these foci the tin form-

basal cells (Fig. 4) while in the other squamous differentiation and kera

CAECINOGENESIS IN SKIN OE HEDGEHOG

213

ation was evident (Kg. 5). Nnmeioos melanin-containing phagocytes were present
1 -T 1 thp Ivmtih node but no melanocytes were detected.

The^SriTof the lesions seen in this hedgehog were type III kerato-
acanthomas (GhadiaUy, 1958, 1959). Many stages of evolution of this lesion were
detected The mature deeply placed herry-shaped lesion is shmvn m Fig. •
Lgressing lesions with marked fibrosis and disintegrating epithelial of

pse?do-carcinomatous appearance, strikingly simdar to f ™

acanthomas of man and other experimental animals were also found. It has been
claimed that the type III kerato-acanthoma probably arises from the hair germ,
or from the deeper part of the hair follicle which is developed afresh at each hair
growth cycle from the hair germ (Whiteley, 1957 ; Ghadially, 1958). Hitherto,
morphological eifidence supporting such a concept has been lacking primarily
because a small early lesion starting at the base of the follicle cannot be deleted
early enough macroscopically in the skin of the experimental animals. Kg. /
and 8 show a deeply placed cystic epithelial lesion which was discovered acci-
dentally. It is almost certainly an early stage development of a type III kerato-
acanthoma. Whiteley (1957) found that in the rabbit type III kerato-acanthomas
developed during the resting phase (telogen) of the hair growth cycle. The same
seems to be true in the case of this hedgehog for the spine follicles in the surrounding
skin were in telogen.

niscnssiox

The significant fact that emerges from this study of hedgehog tumours is that
in spite of such marked obvious morphological differences between the skin of
this animal and that of laboratory rodents and man, the types of benign epithelial
tumours produced by carcinogenic action are essentially similar in all these
species. It has sometimes been argued that the results of carcinogenic studies in
rodent skin thickly covered by fur can hardly be applicable to the skin of man.
Whiteley (1957) and Ghadially (1958, 1959) have already demonstrated the
astonishing similarly of both behaviour and morphology of the kerato-acanthomas
of man and experimentally produced tumours in rabbit, mouse and hamster skin
and now we observe that the same is also true in the case of an insectivore — the
hedgehog. Kerato-aeanthomas have also been produced on the feather-bearing
.skin of birds (Rigdon, 1959). The fact that morphologically similar tumours
occur in such a vast variety of species with different types of hair follicles and their
homologues does not detract from the argument that kerato-aeanthomas arise
from these structures, for it has been shomi that when the glabrous skin of the
duck s foot (Rigdon, 1956) or the exteriorised cheek pouch epithelium of the
hamster (Ghadially and Illman ,1960) is painted with carcinogen many papillomas
hut no kerato-acanthomas are produced. ^

of development of a type

ini, t E’^Pe™ental evidence derived from the stSy

moThoIop'S

rodents led Ghadially (1958 1959 ^ m ^ ™ laboratory

wwcl, 1 , 0 . oortai..,,. orf.on i„ « fashion. l

214

F- GHADIALLY

that th)s tumour has arisen from the epidermis or from the sunerficial uarh nfu
spine folJicIe, for serial sections have demonstrated no point of contaJ^i. betireen
these structures and tJie tumour. The position vdiich this tumour occupies, under
tlie quiescent spine foJJieJe and the erector spinal muscle is consistent Vith the
])osition where one would expect a tumour from the spine germ to lie (The hair
germ or the spine germ and the deeper part of the follicle which is developed from
«iis stru cture he below the plane of attachment of the erector muscle of the follicle )
this lesion was found in an animal in which many t 3 q 3 e III kerato-acaiitlioiiias
developed. It is therefore not surprising that an early lesion was also found in
tin's animal. However since this tumour was found in an animal which also
had a carcinoma it can be argued that we are here witnessing an infiltrative or meta-
static extension of this tumour. Tin's seems unlikely for the following reasons.

(а) Serial sections sliorv no connection between this tumour and the carcinoma.

( б ) The lesions produced metastatic squamous carcinoma are usually irregidar
and infiltrative and not rounded and sharply demarcated as the one shovn in
Fig. 7 . (c) This tumour resembles a kerato-acanthoma in ever3’’ detaii, for this is
a C3"stic lesion with a central core of keratin surrounded b3’' irregular squamous
epithelium shomiig pseudo infiltration. The characteristic ]3'’mphoc3iiic inffltration
is also seen at the periplier3' of tin's tumour.

SUMMARY

It has been shoum that tumours can be produced in the skin of the hedgehog,
wliich is an insectivore, b3'- repeated applications of DMBA.

Man}' kerato-acanthomas, a papilloma, and a carcinoma with secondar)’
deposits in a l3’’niph node were produced.

In spite of the marked morphological differences that exist between the skin
of the liedgehog and the skhi of man and laboratory rodents the tumours that
arise show a marked morphological similarit}'.

An earl}' stage of development of a type III kerato-acanthoma was found ami
studied b}' serial sectioning. The appearances seen support the concept of Bdiitelc}'
and Ghadially that these tumours arise from the hair germ, or the deeper part ot
the hair follicle.

EXPLANATION OF PLATES

Fio. 1.— Tumours produced on the dorsal skin of a hedgehog by 9 : 10 dimethyl 1 : 2 benznn-

tliracene. X j.

Fig. 2 . — ^Papilloma, x 24 .

fJo.' I’— Secondary de^sit in Ijunph node shmving basal-ceU

Fio. 5.-Secondao^ deposit in lymph node shoivmg squamous-ceU d.fferent.ation.

Fig 6. — A portion of a mature III kerato-acanthoma. ^ . This is a c3’Stic

Fm. 7.-Fa% stage of development of « ^

tumour with a central mass of keratin. The tumour is seen

spine foUicle and the erector spinae muscle. X 14. central keratin-fJied

s.-High power from cystic wall of turnour Jean se^Lratin ma.«cs.

cavity lies to the bottom of the picture. ™ .L periphery of the tumour,

neoplastic squamous cells, and Ij-mphoejdic infiltration at the perip. er>

Xl35.

BkitISH JOtTEXAI. or Ca>'CEB.

Vol. XIV, Xo. 2.

CrhadiaUy.

CARCINOGENESIS IN SKIN OF HEDGEHOG

215

I am indebted to Professor D. H. Collins for helpful advice and critieism and
to Sir. J. H. Kugler, T. L. Platts, A SSTiitaker, J. N. Carver and S. Wall for tech-
nical assistance. The ivork was supported by grants from the Universitj’^ of SheflSeld
Sledical Research Fund and from the British Empire Cancer Campaign.

REFERENCES

Ghadially, F. N. — (1958) J. Path. Pact., 75, 441. — (1959) Ibid., 77, 277.

Idem ANB Iloiak, 0. — (1960) Ibid. In press.

Rigdon, R. H. — (1956) Cancer Ees., 16, 804. — (1959) Arch Derm. Syph., N.Y., 79, 139.
Whiteley, H. j.— (1957) Brit. J. Cancer, 11, 196.

21G

fluorescein-globulin affinities of thf

SHORE PAPILLOJIA

C. J. LOUIS

/'row; f/ic Dcparlmenf of Pathology, University of Melbourne, Melbourne, AjislraUa

Received for publication April 30, 1960

liiE fluorescein-globulin method of staining which distinguishes neoplastic
liom non -neoplastic cells has now been emploj'^ed in a sufficiently large number of
cases to demonstrate that it applies consistently both to chemicallj^ induced
tumours in animals and to naturallj’- occurring tumours in both animals and man
(Louis, 195 ti, I95Sa, 19586; Louis and Varasdi, I960). The test also affords
a means of differentiating acute leukaemia from the chronic form {Louis, 1957c,
1958d).

It has been shonui that all normal tissue ceils of vertebrates and some inverte-
brates stain well and fluoresce brightly provided they contain an adequate amount
of cj^oplasm. The few exceptions that do not stain, namely : resting connective
tissue cells, red blood cells, cells of the central nervous system and cells in tissue
culture, have been discussed previously (Louis, 1958c; Louis and White, 1960).
The significance of the staining reaction although not yet completelj'^ understoo(!
has been attributed to protein-protein interactions between the basic cj^oplasmic
protein (s) of the normal tissue cells and the labelled serum proteins which have
been rendered less basic by the conjugation process (Creech and Jones, 1941fl,
19416 ; Hopkins and W^ormall, 1933), Tlmt no serological imph'cation is concerned
in this reaction has been demonstrated bj^ the observation that identical results
could be obtained by using normal rabbit globulin (Hughes, Louis, Dineeii and
Speetor, 1957), albumin and globulin fractions of many different species and even
of the same animal (ICing, Hughes and Louis, 1958, 1959) and finally by egg
albumen (Hughes and Louis, 1959). AH malignant cells, on the other hand, even
though containing large volumes of c 5 'toplasm, failed to stain. Concomitant vit i
the onset of the malignant change tumour endoplasmic proteins become kfS
basic (Eldredge and Luck, 1952; de Lamirande, Allard and Cantero, lOa ,
Sorof and Cohen, 1951). Hence it is to be expected that these proteins would line
a decreased capaeity to form complexes with the fluorescein-globulin conjuga ft.
This has been found to be the case in a large number of tumours examined, n
addition, certain presumably “ premalignant ” but morphological^ norma ce »

were found not to stain. j

Because these observations relate only to chemically induced and na i •
occurring tumours it was decided to investigate a virus induced tumour, par ^
study virus infected cells and partly to see ff this tj'pe of neoplastic chaise i -
from the chemicallj'- induced variety. Thus the results of the staining a ni
the Shope papilloma during its course of development are presented an isc i—

PLUORESCEIN-GLOBULIN AFFINITIES OF SHORE PAPILLOMA

217

METHODS AND MATERIALS

Papilloma virus

The virus for inoculation was supplied by Dr. Richard E. Shops of the Rocke-
feller Institute in tissue slices of freshly removed warts stored in 50 per cent,
dvcerol. The tumour slices were ground with washed sand and normal saline
to yield a 10 per cent suspension which was subsequently filtered through filter
paper (Whatman No. 1). This filtrate was used for inoculation.

Inoculation of rabbits

Twenty-four young white rabbits of mixed breed had small areas shaved on
the dorsum of the distal halves of both ears followed by fight scarification ivith
sand paper. Inoculation was effected in some by rubbing the virus suspension into
these areas, whilst in others by intradermal injection of OT ml.

A second group of 12 rabbits had similar areas of skin prepared which were
painted daily 5 days per week for approximately 12-14 weeks with the following
carcinogens : 3 ; 4-benzpyrene, 1 ; 2 ; 5 ; 6-dibenzanthracene and 20-methyl-

cholanthrene. These carcinogens were made up in 0-2 per cent solution noth
benzene. When small wart-fike projections appeared the carcinogen application
was stopped and each rabbit was given 2*0 ml. of the infected filtrate intravenously.

Production of hepatomata

Adult Sprague Dawley rats weighing 150-200 g. were used. Hepatomata were
induced by feeding 0*06 per cent 4-dimethylaminoazobenzene in a 20 per cent casein
diet containing 2 mg. of riboflavine per kg. Rats were killed under ether anaesthesia
at 2 weeks and then at weekly intervals imtil 12-15 weeks when liver tumours
appeared. These have been described in detail elsewhere (Hughes, 1958; Hughes,
Louis, Dineen and Spector, 1957) and are used here for comparison with the
rabbit tumours (Table I).

Prc])aration of sera

The globulin fraction was extracted from the serum of both inoculated and
non -inoculated rabbits by precipitation rvith half saturated ammonium sulphate
and after dialysis was conjugated rvith fluorescem isocyanate (Isomer I) by the
method of Coons and Kaplan (1950).

Preparation of tissue sections

In most cases small pieces of biopsy material were taken from the margins of
1 0 U nmurs and nnmedmtely snap frozen by dropping in isopentane preioled
to C m^an cthanobdrj- ice mixture. Erom these unfixed frozen sections
were cut at o-/ /; by a method previously described (Louis, 1957a).

I Inorr/^rcncc staining, microscopy and photograjiliy

were I mated with the labelled elobulins fnr in -1 ^led tissue sections
P-«>y in .!„» Cnngcs of buffered suliue, audriiSte;

21S

C. J. LOUIS

jvore feed ;„ ,0 por oenAoLol-sarJ, sttaedwS'ifaell”
or compaijsoii, the same areas ^rere repJiofcographed using viable iiglit.

RESULTS

The macroscopic and microscopic observations on papiJJoinatosis experimeat-
ally induced in this series of animals ivere similar in all cases and good accounts
have been given by Sliope (1933), Rous and Beard (1935) and Sjn^erton (1952).
±or tJie purpose of this study the development of these cutaneous tumoius is
treated in four stages :

(ff) The phase of active epidermal proliferation ;

(A) the quiescent phase ;

(d) the malignant phase.

Unfixed frozen sections prepared from all tissues investigated were examined
first in the unstained state to exclude any intrinsic fluorescence present indepen-
dently of the use of the stain. As rvith prerdous experiments it was observed that
elastic tissue and the ground substance of cartilage emit a bright yellow auto-
fluorescence and keratin pale blue. A particular effort was made to avoid confusing
these with the characteristic green fluorescence which results after staining with
the fluorescein-labelled sera.

Normal rabbit epidermis

All tlie cellular layers of the stratified squamous epitheh'um in rabbit skin
showed a strong affinity for fluorescein-globulin complexes and emitted a bright
green fluorescence in ultra-violet light (Fig. 1 and 2). There was a uniform distri-
bution of the dye Avitliin the cells in which the cytoplasm, but not the nucleus,
fluoresced. The cells lining the hair follicles and sudoriferous glands also stained
uniformly.

Proliferating epidermis

This stage of development became apparent macroscopically between the
lOtli and Mill daj’- after inoculation as small papular vesicles winch ® ^

progressed and developed into large verrucous masses (Fig. 3). This phase laste
approximately 5 months, Mcroscopically, in their lyell developed stage,
showed considerable thickening of the epidermis ivith papillary ou .

the surface and gross keratin formation (Fig. 4). There was active pro i ‘
of the priclde cell layer -with increase in the number of cells and numero
figures. Many of these cells contained pigment. These cells, J®'™'
regular in form and the deeper lajmrs clearly demarcated from ’
connective tissue stroma. Small collections of wandering cells, princ p J
small round cell type, were present in the superficial dermis l,,merulastic

Histochemically, after staining writli_ the J^iformly

cells and those wliich were undergoing mitosis fluoresced brig i ‘
as normal epithelium (Fig. 5 and 6).

TLTJOBESCETX-aLOBTJLIN AFFINITIES OF SHOPE PAPILLOMA

219

Premalignant phase .

About 6 months after inoculation the growths stopped gromng
which was evidenced microscopically by the gradual decrease “
ceUs absence of mitotic figures and desquamation of the superficial epidermal
layers. This persisted for a further 6 months, during which period some mvolution

took -place (Tig. 7 and 8). . . r ^

The fluorescence staining characteristics in the beginnmg of tins phase were

similar to those observed udth normal and hjqierplastic epithelium ^namely,
a uniform staining of the epithelial cells. At the lOth month there were observed
gioups of epithehal cells which showed a diminished affimty for the stain and
failed to fluoresce in ultra--violet fight (Fig. 9 and 10). These islands varied in
size and contained anything from 6 up to 40 cells. No morphological evidence of
malignancy was detected when these sections Avere stained uith a routine stain
such as haematoxjfiin and eosin. Again the cells Avere regular in form throughout
and the basal layers sharply demarcated from the underlying stroma.

Malignant phase

Irregular “ islands of loss ” of staming Avere obsen^ed until 15 months Avithout
histological evidence of any malignant development. At about this time evidence
of invasion became apparent. Masses of cells, arranged in small groups and strands
and shoAsing epidermoid difierentiation, extended into the underlying stroma.
These Avere irregular in shape, size and nuclear densities and showed many mitotic
figures. Initially these Avere confined to the superficial dermis (Fig. 11-14).
Later collections of cells AA'ere seen invading the cartilaginous plate (Fig. 15-18)
and finally penetrated through the ventral aspect of the rabbits’ ears. Here a
clear-cut difference aaus observed between the non-fluorescing invading txunonr
tissue and the brightly fluorescing invaded ventral epidermis (Fig. 19 and 20).

Exposure to chemical carcinogens

In rabbit ears pretreated with carcinogens similar microscopic and macroscopic
changes u ere observed but the malignant transformation after intravenous
injection of the virus occurred much earlier than in the first group. The epidermal
cells shoAved strong affinities for both conjugated dyes up to the 6th month stave
of development Avlieii islands of loss of staining could be demonstrated. These
islands persisted only for 3 months before obvious malignant changes became

The resultant tumours appeared more active than the previous ones in that
mvmsion Avas much more rapid and metastases more AA-idespread.

^)|ctions taken from both primary^ and secondary- nodules no fluorescence
was emitted by the tumour cell cjfloplasm. nuorescence

lint hvrr after i-dimethjlaminoazobenzene administration

^'■‘^'""'nation ofsections from the fivers of rats fed 4 rUmo+u i • 7

shoAved that all the parencliATual cells stainpd „r7,-r ^ ^™®^^y^ammoazobenzene

p^hymYreTS^

„-„e„ “JS

220

C. J. LOUIS

failed to stain ivitli tlie labelled sera
normal liA^er cells.

m contrast to the brightty fluorescing adjacent

UISCUSSIOX

Although numerous studies on the nature of neoplasia have been marie anrl
much mformation collected, an understanding of the fundamental nrorp'<;<;e’
involved m still distant. During the last center numerous vague LSSeS
ftctois AACie held responsible for the neoplastic change but these have recently
^ distinct agents, which have been subdivided

viruses) ’ elated rays) and organismal (particularly the

Jluch of our present knowledge of carcinogenesis has stemmed from the dis-
covery and study of tlie chemical carcinogens. To date many such substances
iia\ e been discovered but it lias been sliov^n that often a carcinogen Arill affect
only a particular organ of a particular species. TJiis effect may be produced by
local apjdication, oral administration or b}’’ injection parentall}'. Repeated
observations with three groups of substances, namely : aminoazo dyes (llillcr
and Jliller, 1947), ai'omatic polycyclic hy'^di'ccarbons (Heidelberger and Molden-
hauer, 1957 ; Heidelberger and Weiss, 1951) and aminofluorenes (Jliller and Miller,
1952), have shown tliat the carcinogen is taken up by the cells of the susceptible
tissue. Differential high speed centrifugation of the tissue homogenates and subse-
quent electrophoresis (Eldredge and Luck, 1952 ; de Lamirande, Allard and Can-
tero, 1953 ; iSorof and Cohen, 1951) indicated that the carcinogen became firmly
bound to a imotein or protein complex present in the soluble fraction of the ceil
cytojflasm. Investigation of the tumours which developed subsequently showed
that the tumour cells not only lacked this djm but also the protein(s) which was
present in tire parent cell and to which the carcinogen presumabty became attached
(I\Iiller and IMiller, 1947). Thus there was a clearly defined and easily reproducible
cbfference between a normal and a malignant cell. This difference has been
demonstrated histochemicallj^ in a variety of naturally occurring and chemically
induced tumours using fluorescein-globulin conjugates (Louis, 19576 ; Louis,
1958a ; Louis, 195S6 ; Louis and Varasdi, 1960). The normal cells, which con-
tained the basic protein(s), reacted with the labelled djm and emitted a bright
green fluorescence in ultra-^^olet light rvhereas the malignant counterparts dit
not. In most sections prepared from the livers of rats fed 4-dimethylamiiio-
azobenzene non-staining islands of cells were seen. The failure of tliese islanrs o
morphologically normal Imt perhaps premalignant parenchymal cells to fluoresce
appeared to be of great aetiological significance.

Our knoudedge of virus carcinogenesis, on the other hand, is relativeh meag ■
Difficulties encountered in extraction and purification of these viruses are
sarily responsible for the poor understanding of this group of neoplasms, nice
Shope papilloma virus was readily available and provided a good examp e o
relationship of viral agents to benign and malignant growths it lias been s u
detail in the present paper. Tliis virus, like the chenaical carcinogens, i< . ^
slioivn to possess strong cytotropism causing only^ epidermal cel s o ra
proliferate and, after a latent period, to become malignant (bhope, ' j
present investigation examination of maii}’^ examples yd;, Lnii"

which had progressed to the tivelve month stage of developmen si i .n,nj
affinities for the conjugated dyes and fluoresced as brightly as the a ja

pludbesceik-globulin affixities of SHOPE PAPILBOMA

221

fafled to fluoresce. Tlfls was particularly striking in the early stages u here im asion
was confined to the superficial dermis. Here groups of cells showing g«o J^qua-
moid differentiation M-ith Avell-developed keratin nests and intercellular ]iriclJc.s
lacked all affinities for the fluorescent stain and failed to show a positu e staining
reaction (Fig. 11-18). Here also islands of monihologically normal epidermal
cells have been observed which have shown the characteristic loss of staining
(Fig. 7-10). The persistence of these islands, their possession of fluore.sceiice
staining characteristics similar to those of frank ep'dermoid carcinoma cells aiul
the subsequent development of carcinoma at these sites suggests stronglj' that
tliey are premalignant foci.

In all tumours, both experimentally induced and naturally occurring, examined
by this method of study, the difference from normal tissue has been clear-cut
and Avell defined. Since this difference appears to be due to the absence from the
malignant cell of a protein complex, it seems probable that such a change would
not necessarily he an abrupt one. Indeed, the present and previous investigations
indicate that the final form of tumours is due to a series of changes. The natvirc
of such changes occurring in the cells during the preneo]flastic phase and con-
comitant with the development of malignanc}' is still unknomi. The first histo-
cheniical evidence that a change has occurred is indicated the appearance of
persi.stciit i.slands of morphologically normal cells with a diminished affinity for
fluorescein-globulin complexes, a feature common to both chemically and viral
induced tumours. Thus if the changes occuiTing during viral carcinogene.sis
are compared u-ith those induced with a chemical carcinogen such as the aminoazo
dyes (I’ahle I) the changes leading to malignancy are found to be similar. Further-
more, the Shope virus can act sjniergistically with certain carcinogenic hydro-
carbons (Rogers and Rous, 1951). In the present investigation, this phenomenon
has been demonstrated nith 20-methylcholanthrene, 3 : 4-benzpjTene and
1 •. -I •, 5 ; G-dibenzanthracene. Intravenous injection of papilloma virus induces
malignant tumoims in areas of skin nhich have previouslj'’ been treated AA-ith

'I A r.LU I . — Com pariaon
Hepatomnia

of Fluorescence Staining of Shope Rabbit Tumours and
induced in Rat-s by i-Rimethylaminoazobenzene

SjM'fifS sj^coifirity
<>raaii bpfcificity

of cnrciiiopon in
uftrinul i-fIU

MuikIx <,f lo-s of Hiiorc-.s.

Miiiimif; ■' of inor-
pUolojjirnlly innocoiu

I’ri'-i'iuv of nm-inopon in
tmiifjur

Shope virus

Rabbit

Epidennis

Can be extracted from tumours
up to 9 months after inocu-
lation

Detected beUvecn 12th-1.5th
month after inoculation and
before development of frank
malignancy

Jialignant change occurs at ap-
proximately IS months. Xo
vini.s extracted from tumour
Ussuc. Tumour cells fail to
fluoresce

4-DAB

Rat.

Ln’cr.

Fiiwly bound to soluble cyto-
plasmic proteins for 9-12
weel^. Tissue turns pink on
acidification.

Detected 4—12 Aveeks after
feeding 4-DAB.

Hepatomas appear at approxi-
mately 12 weeks. Tumour
tissue does not turn pink on
acidification. Tumour cells
do not fluoresce.

222

C. J. LOUIS

polj'cyclic hydrocarbons. In tliese cases the tumours appear sooner, they are
larger and metastasize more readily^. Although tumours induced in this maimer
are similar to those induced with simple virus inoculations, Smith, Kid and Eons
(1952) were not able to extract an infective filtrate from these. However, in spite
of such similarities there niaj^ be a distinct difference beWeen the presumed loss
of a dye binding cytoplasmic protein and the situation in which there is failure
to extract an exogenous virus-like agent from a neoplasm.

In resume, therefore, it may be said that the fluorescein-globulin affinities of
normal, hyjierplastie and neoplastic cells of a virus induced papilloma areidentic.il
to tJiose of cliemically induced and naturally'' occurring neoplasms. The distinctioii

EXPLANATION OF PLATES

FlO.. 1. — Rnbbit .skin. Unfi.vod frozen section stained with fluorescem-globulin complex' and
allowing bright uniform fiuorosceneo of cytoplasm of epithelial cells ; vacuoles and nuclei
do not .stain, x 120. . . r i ?•

FlCi. 2. — Same section and area as shown in Fig. 1 after fixation in 10 per cent formol-saline
and .stained with hnemafo.vylin and eosin for comparison. X 120.

Fio. 3. — Rabbit ear showing young papilloma (3 months after inoculation). X3.

Fio. 4.— Paraffin section from tumour in Fig. 3 showing the topical structure of benign pajiii-

loinn. Haomatoxj-lm and eosin. x30. j -m a,

Fic. .'i.—Unfixed frozen section prepared from tumour m Fig. 3 and storoed with ttaoresceui.

globulin complex. The cytojilasm of epithelial cells stains uniforrfy. X 2^.

Fici, (iv — -Snino section and area as sho'H'n in Fig. 5 subsequently fixed in fo
stained with iinomatoxylin and eosin for comparison. X220.

Fio. 7. — Shone papilloma 10 months after inoculation, xl’5. k

Fio. S.— Paraffin section from tumour in Fig. 7 showing 2™ atoxvlia

regular in form througliout and basal layers clearly demarcated from dermis. Haemaf . ,

Fim ll.^-^Unfixvd^Lzon section prepared from margin "how^'ZiMi^

fluorescein-globulin comple.x. loss >>). x240.

fluorescence and some complete absence of ^orMcence ( i ^ j m pgj. cent formol-
Fio. lO.-Same section and area as shown in Fig. 9 subsequently m ^0 p®® ^
saline and stained with haematoxylin and eosm for comparison. Eote that

cing cells are morphoJogically innocent. X breakdown of surface keratin

Fio. II.— Shope papilloraa 15 months after inoculation showmg DreaKuo

Fio”" l-Cffintction from tumour in Fig. U. There is earl^'.invasionrf dermis by well

Fic^ —Sniiie seetion and nren as slio.™ in Fi^ 'for comparison. "rhe non-Cresemg ooiis-
saline and stained with haemotoxylm and x240. ^ ,

although irregular in form, show good ® showing extensive ulceration. X 1.

Fio. I5.-^Shope papilloma 18 months are seen invading through

jZjg, J6. — paraffin section from tumour in Fig. 1 ■ P ■ u ■ ht

to cartilaginous plate. x30. Fluorescence photomicrograph showing "g

Fig. 17.— The rectangular area m cartflagLnd outlines of non-finarewmg

autofluorescence (yellow) of ground substance ot g

cells in the background. X -.40. 17 rubseauently fixed in formol-sahne and s <

Fio. 18.— Same section and area as m ®“^®|S"''collections of non-fluorescing cell,

with haemato.xylin and eosin for .^micro-

the characteristic neoplastic tissue. X 240, i„oculatiou. Fluorescence

F.; »«!»,. .«/ .« .. » zi,- « X

.h.

British Journal of Cakcer,

Vol. XIV, Xo. 2.

Louis,

British Journal of Cancer.

Vol. X^\^ No. 2.

Louis.

■av;

British Journal of Cancer.

Vol. XIV, No. 2.

Louis.

223

PLUOEESCEES'-GLOBULIIs AFFINITIBS op shope papilloma

between viral and chemical carcinogens is becoming progressively more difficult
to maintain and the present work supports the view that the virus is a Tenant of
the chemical carcinogen rather than a qualitatively distinct agent, thus the
general fluorescence staining characteristics of malignant cells are remarkably
constant and the results appear independent of any aetiological agent.

REFERENCES

Coons, A. H. and Kaplan, M. H. — (1950) J. exp. Med., 91, 1.

Creech, H. .J. and Jones, R. N.— (1941a) J. Amer. chem. Soc., 63, 1661. — (19416) Ibid. 63,
1670.

Dineen, j. K. and Ada, G. L. — (1957) Nature, Land., 180, 1284.

Eldredge, N. T. and Luck, J. M. — (1952) Cancer Res., 12, 801.

Heidelberger, C. and Moldenhauer, M. G. — (1957) Ibid., 16, 442.

Idem AND Weiss, S. M.— (1951) Ibid., 11, 885.

Hopkins, S. J. and Wobjlall, A. — (1933) Biochem. -J., 27, 740.

Hughes, P. E. — (1958) Cancer Res., 18, 426.

Idem AND Louis, C. J. — (1959) Arch. Path., 68, 508.

lidem, Dineen, J. K. and Spector, W. G. — (1957) Nature, Land., 180, 289.

King, E. S. J., Hughes, P. E. and Louis, C. J. — (1958) Brit. Cancer, 12, 5.— (1959)
Cancer, 12, 741.

De Lamirande, G., Allard, C. and Cantero, A. — (1953) Ibid., 6, 179.

Louis, C. J.— (1957a) Stain Tech., 32, 279.— (19576) Aust. N.Z. J. Surg., 27, 146.—
(1957c) Aust. Ann. Med., 6, 300.— (1957d) Ibid., 6, 277.— (1958a) Brit. J. Surg.,
46, 147.— (19586) Surg. Gynec. Obstet., 107, 317.— (1958c) Brit. J. Cancer, 12,
537. — (19.58(1) Aust. Ann. Med., 7, 219.

Idem AND Varasdi, G.— (1960) Ann. Surg. (in press).

Idem AND White, J.— (1960) Lab. Invest., 9, 273.

Mn.LER, E. C. AND I^Iiller, j. a.— (1947) Cancer Res., 7, 468

Miller, J. A. and JIiller, E. C.— (1952) Ibid., 12, 283

Rogers, S. ,vnd Rous, P.— (1951) ./. exp. Med., 93, 459

Rous, P AND Beard, J. W.-(1935) Proc. Soc. exp. Biol. N.Y., 32, 578

Shopl, R. E. — (1933) J. exp. Med., 58, 607

SoRoF, S. AND Cohen, P. P.-(1951) Cancer Res., 11, 376.

Smith. H . L„ Kidd, J. G. and Rous, P.— (1952) J exv Med 95 PQQ
SvvERTON, J. T.-(1952) Ann. N. Y. Acad, ki., 54, iS.

224

R>U3I0ACTiyE TRACER STUDIES OF RED CELL DFSTRTTrTTnv
IE RATS BEARING A TRANSPLANTABLE TIDIOUR

E. H. BELCHER and SHIRLEY M. SBIPSOY
From the Posijradmie Medical School, London, ir.l2

Received for publication April 2, 1960

An AK.MiA IS frequently observed in cancer patients and also in animals bearing
transiiiantabic or induced tumours ; tliis association is the subject of a recent
review by Price and Greenfield (1958). It is probable that both decreased erv-
throiioiesis and increased erythroc 3 de destruction contribute to the reduction in
liacrnoglobin concentration, haematocrit value or red cell volume observed in
anaemias associated with cancer. However, evidence has accumulated that an in-
creased rate of destruction of the circulating red cells is common in human cancer,
riius Berlin (1951) demonstrated bA”^ the Ashbj- differential agglutination technique
an increased rate of destruction of transfused normal cells in patients with myeloid
or lymphatic leukaemia. An increased rate of destruction of the patient’s own
circulating red colls labelled with glycine-2-^'*C was found by Berlin, LavTcncc
and Lee (1954) in leukaemia. Hyman, Gellhorn and Harvey (1956). using both
the Ashby technique and the transfusion of cells labelled with °'Cr found an
increased rate of destruction of cells transfused from normal donors to patients
with carcinomata and other neoplasms, avhereas cells transfused from cancer
jiaticnts to normal volunteers had a normal or near-normal life span. Hence they
argued the absence of any’ intrinsic defect in the red cells of cancer patients.
]\Iiller, Chodos, Emerson and Ross (1956) also demonstrated bj’ the Ashby and
the “'Cr-techniques an increased rate of destruction of red cells transfused from
normal subjects to patients v’ith cancer of various sites or autotransfused to such
patients.

The results of studies on animals bearing transplantable or induced tumoim
have been more conflicting. Ultnian, Fish and Hy’iuan (1956), in studies of^’Cr-
survival in rabbits bearing the Brow’ii-Pearce or XV2 carcinoma, failed to reieo
any’ increased destruction of red cells except in animals with severe infectiorr
Ehrenstein (1958) used gly’cine-2-^'’C to study’ the life span of the circulating rni
cells in mice bearing the Elirlich ascites carcinoma and found increased rec ce
destruction of the random tyqie in tumour-bearing animals. Greenfield,
and Price (1958), using “sPe-labelled cells, also observed increased red cel dcstra -
tion in rats bearing Ly’inphosarcoma 2788, Hepatoma 3683, the Yoshida asci -
tumour and the Harderian gland Cai’cinoma 2226. They found that as
destruction proceeded, ®*’Fe accumulated Avithin the tumour. In a o
tumours, haemorrhage or thrombosis AA’as observed. In the mouse 'j • ji

HE 8971 and Hepatoma 134 (ascites) and the rat Fibrosarcoma
there was little or no haemorrhage or tlirombosis, anaemia and loss o <
red cells was small and did not occur until the late Loccl

further studies by the same group, Greenfield, Sterling and P I ‘
siCr-labelled red cells to rats bearing the Lymphosarcoma K 2/8S ana

225

EED CELL DESTPvUCTIOls Us TUMOUR BEARENTG EATS

to recover from the tumour nearly all the «Cr lost from the blood ; they concluded
that increased red cell destruction was the consequence of vascular m t v

neighbourhood of the tumour. Price, Greenfield, Sterlmg and IMacGardle 19o9)
traSfused mixtures of “‘Cr- and ssre-labelled red cells to rats vnth Lymphosar-
coma R 2788 and found that the subsequent distributions of the two isotopes
vdthin the tumour were similar, uptake being greatest m haemorrhagic area^ less
in necrotic areas and almost neghgible in white suable tumour In this tumour,
haemorrhage into the surrounding connective tissue is an important phenomenon.
The extent of increased red cell destruction in other tumours could be correlated
with their histological appearance. Thus in Fibrosarcoma 4956, little haemorrhage
was observed and increased red cell destruction was not detected until the tumour
had become massive and ulceration of the skin had occurred.

In the investigations described below, red cell destruction has been studied
by the “iCr- and ssFe-tecliniques in rats of the “ August ” strain bearing the
transplantable mammarj'^ adenocarcinoma R 2426, in which neither haemorrhage
nor necrosis are observed. Particular interest was attached to this tumour since
a haemolj'tic episode in the host animal had been observed to follow its implanta-
tion. Preliminarj^ reports of these studies have aheady been pubhshed (Belcher,
19.58, 19.59).

MATEKIALS AXD METHODS

Rats. — Rats of the pure-line “ August ” strain of weight 100-200 g. were used
tlirougliout. They were maintained on Medical Pvesearch Council Diet No. 41 and
water ad libitum. Frequent observations on splenectomised animals failed to
reveal any latent Bartonella imcris infection.

Transplantation of tumour. — Tumour R 2426 was transplanted at intervals of
about 4 weeks. Implantation of tumour was carried subcutaneously under aseptic
conditions through a trochar mserted into the left abdominal wall, the site of
implantation being afterwards closed by a small suture clip. Four weeks after
implantation, vhen transplantation was again due, the growing tumour was firm
and round with a maximum diameter of about 2 cm. On sectioning it was
normally found to be white and viable throughout, rdth no evidence of necrotic
or haemorrhagic areas.

Sludirs irith •’'('r-JabcUed red cells

wJrii ®'Cr were made as described by Belcher and

Harri.ss (1 ).,<)) In order to reduce the possibility of uncontrolled transfusion

v'iVl^^Kv'lsSotrr'o ’ofblood nte^-ttd”" ™

.\vnngo containing ()•! ml. heparin (“ Pullrin ” car^ac puncture into a

ami delivered into a graduated^est tube One ml’ Medical Supplies Ltd.),

> nd. of a solufion of socSi

about .)(! //c./ml. ^'Cr (specific activitv obn, f sahne containing

"iixtun> was allowed to stand for .30 nfinutes with oS

gently eentrifugetl. the supernatant diluted shaking. It was then

"■ '-oiui s

22G

E. H. BELCHER AND SHIRLEY M. SIMPSON

labelled coll suspension was further diluted with isotonic saline if desired
added s’Cr, 90-95 per cent entered the red cells.

rlolnl?- suspension 0-5 ml. in volume were injected vithoiit

dclaj into litter mate recipients, injection being carried out either intrarenoudv
into a lateral tail vein or intrapcritoneally. At daily intervals thereafter, thee.v
tronie tip of the tail of each recipient was cut under light ether anaethesia and a
0-0- nil. blood sample taken into a haemoglobin pipette and delivered into 5 ml.
aminoniated distilled water for haemoglobin estimation. Haemoglobin was
estimated colorimetrically as HbOa the haemolysed blood samples. These were
then transferred to ® inch diameter sample tubes and assayed for siQr in a well
scintillation counter coupled to a scaler through a linear amplifier and single
channel jiulse height discriminator/analyser. The ^^Or concentrations in successive
samjiles of blood were e.xprcssed as percentages of that of the sample taken from
the same animal on the first daj'^ after transfusion and the result corrected for
the increase in blood volume with increase in weight of the animal during the
period of observation (Belcher and Harriss, 1957).

Studies of ®'Cr-distribution in the tissues were made as described by Belcher,
Lamcrton and Harriss (1958). Recipient animals were killed by cervical fractwc
under ether anaesthesia. They'^ were then exsanguinated as completely as possible
by ojiening the thorax, excising the left lung and injecting isotonic saline into the
left ventricle of the heart, diluted blood being removed as it accumulated in the
thoracic cavity. The carcasses were then carefully dissected and selected tissues
removed to f inch diameter sample tubes for ®'Cr-assay. Small corrections were
ajiiilied for the variation in efficiency^ of the scintillation counter with sample
volume.

Studies with ^^Fe-labeUed red cells

Red cell survival studies with ^’I’e were made as described by Belcher ami
Harriss, 1959. The circulating red cells of donor animals were labelled in nio
with ®®re as follows ; 10 /re. ®®Fe (specific activity 1-5 /m.//(g. Fe) as ferric ch on c
in 0-5 ml. 1 per cent w/v sodium citrate solution were injected intravenous y o
each donor. After an interval of 3 daym to allow cells labelled in the erythropoic ic
tissues to emerge into the circulation, about 2 ml. of blood were withdrawal
cardiac puncture from each donor into a heparinised syrringe and dehyere m o
graduated test tube. The sfRe-labelled red cells were further labelled m w''"
5iCr as described above if desired. Portions of labelled cell suspension O'o n
volume were then injected intravenously into litter mate recipients. Bai y " ,
blood samples were taken from the cut tails of the recijiient anima sj*® ^
described, haemolymed in 5 ml. ammoniated distilled water an ™ „ pf

I inch diameter sample tubes for radioactive assay. ^ To suppress re-u _
s®Fe from destroyed red cells by the erydliropoietic tissues Uenfcrs

0-1 ml./lOO g. body weight of an iron-dextran complex (

Laboratories Ltd.) containing 50 mg./ml. Fe were given mtramusculc y
out studies of red cell survival of ®®Fe-]abelled cells. .

In the radioactive assay of samples in single tracer experimen
ssFe alone, the discriminator /analyser unit was operated as a simpi ] fyj

In double tracer experiments with «Cr and s®Fe, samples wmefimt ass^y^
®8Fe by using the unit as a discriminator set at a level such th . •jirY „sing
almost completely insensitive to s^Cr. Samples w^ere then assay

227

PvED CELL EESTRECTIOK EC TUMOUR BEABECG RATS

the unit as an analyser aligned on the peak of the gamma spectrum, a cor-
for the small contribution to the total eountmg rate due to
s>Ee on the bafis of the already determined "Fe content of the sample.

EESTjLTS

Eesponse of “ August ” rats to implantation of tumour R 2426

August rats receiving subcutaneous implants of tumour R 2426 undergo an
acute haemolytic episode between 10 and 15 days after imp antation, at lylnch
time the growing tumour has the size of a small pea. Durmg the episode, which is

accompanied liy haemoglobmuria and which may be fatal, the haemoglobin
concentration of the circulating blood falls to about 5 g./lOO ml. (Fig. 1) If the

concentration returns to normal ivithin a further

ttc Mehcd

Uutiocr impisni. Ihc fate of tlie eirculatinnrpd animal n'hich is then given a
can be folimvcil (Pig. 2). Up to the start ofthe^h.' ^ Itaemolytic episode

IS witliin the normal limit.s quoted bv Belcher anr^H^^^ episode, red cell survival
episode almost all of the transfu^ed'^celk are rt ^ (19o9), but during the

ibe I, loot! falling more .steeply than the haemoT “^Gr-concentration in

come «C„, ^

228

E. H. BELCHEB AND SHIRLEY M. SIMPSOK

implantation with tumour R 2426.

r7 Range of ®'Cr-coneentration in normal “ August ’ ’ rats transfused with 5>Cr-!nbelled

cells from litter-mate donors.

transfused to tumour-bearing recipients which have already suffered their hacnio
]3rtic episode and are in recover}'- from it, the labelled cells are not rapidlj' reniotet*.
red cell destruction being only slightly, albeit significantlj'-, more rapid lah
normal animals (Kg. 4). At tliis time therefore, it appears either that the
tration of the haemolytic agent in the host animal is reduced or that some mcc i.
ism is operating to reduce its effect on the circulating red cells.

* In this and subsequent figures, except Rig. 11, the graph lines shoivn by solid and open P
refer to two identically treated rats.

229

MD CELI. BESTSOOTIOK IN TraiOTO EEABING KATS

oI ce,Ufr.n ^ "

L- - — ■ j ^^•'>"P<‘ of' ‘’Cr-concetitra tion in nonnal ‘■- August " rats transfused -n-ith ^'Cr-lahpIIprT
froirv littor«!U5'\tc donors.

W 1r II cdls from tmiiour-bearmg ammals in recoverj' from their haemoh-tic enicmrle
are labelled and transfused to other tumour-bearing animals in Tecorlrv rSl eeb
destruction aiipears within normal limits (Fia. 5) However in tbi«
transfused cells have a grossly abnonnal aU distribntil Sn ^ situation the
vounu cells, .since mo.st^f the donorTcirc4t“^^^^^^^

.1,0 C,„,„ ,„o« oirc„„,s,.„c«, the obsetfation o? :ptaSSty

230

E. H, BELCHER AND SHIRLEY M. SBIPSON

observed wlien destruction rate, such as is

]>ionts ill recovery. ^ normal donors to tumour-bearing red-

Fig. 4. — Blood haemoglobin and ^’Cr-concentration in tumour -bearing “ August ” rats
transfused with 0-5 ml. ®*Cr-Iabelled red cells from normal litter-mnte donors 14 days after
implantation u-ith tumour R 2426.

xzzzm Range of “tCr-concentration in normal “ August ” rats transfused with ^'Cr-labelled

cells from litter-mnte donors.

Survival of red cells from tumour-bearing donors doubly labelled with and
and transfused to tumour-bearing recipients
It is of interest whether red cells are destroyed during the haemolj'tic
in a random fashion udthout regard to age, or whether older cells are morerapit^.^
destroyed than younger ones. To study this question, a donor rat ^ Three

tumour 3 days previously was given an intravenous injection of 10 /te. r e.
days later, when the ^^jPe-labelled red cell precursors had emerged into the cir

bed CEtt DESTEUCTIOK K rOMOOE BEABIKO BATS

231

lating blood, blood

and labelled m vitro \nth. • _ included a single cohort of young

1 Himp:i' of ‘'Cr-courcntration in normal “ August ”

colK friiin liUor-iiinto donors.

rats transfused with ='Cr-labelled

t.u.-m..;WA, »f ml cdl imcmon h tW the

.■«n... f:„. ,.... d„cs Ita J.CV. Apart

2312

E. H. BELCHER AND SHIRLEY M. SIMPSON

<l-t.-oyod at random, rrithont regarii age.Trt

Fig 6. — Blood hnemoglobin, and ®®Fe levels in tumour-bearing “August” rats given
0 • 5 ml. of red cells doubly labelled with ®*Cr and ^®Fe from tumour-bearing litter-mate donor
6 dn 3 's after implantation with tumour R 2426.

O O ®®Fe content. O O ®*Cr content.

Range of ^^Cr-concentration in normal “ August ” rats transfused with ^*Cr-IabeIIcd

cells from litter-mate donors.

Sites of destruction of red cells from tumour-bearing donors labelled until ‘’'Ci' avA
transfused to tumour-bearing recipients
To identify the chief sites of red cell destruction during tlie iiaemol}^ ic epbwlCt
red cells from tumour-bearing donors were labelled nuth “'Cr and
tumour-bearing recipients before the development of baemol3’'sis. During or a f
the subsequent episode, recipient animals were sacrificed and exsanginna

bed cell DESTBUCTION IX TUMOUB BEABIXG EATS

233

During or After Subsequent Haemohjhc Episode.

Inter\'al betiveen implantation
and transfusion (days)
Interr-al between implantation
and assay (days)

Tissue

^'Cr content

‘‘Cr content

% injected dose)

^ '

injected dose)
-f ,

f ’

18-0

17-0

13-0

14-4

15-0

10-0

12-0

13-5

27-0

23-0

12-0

27-0

.5-0

2-5

1-5

5 • 5

1-.5

0-8

0-4

1-0

0-1

0-2

0-1

17-3

17-8

5-4

8-0

4-5

14-0

25-0

23-7

11-0

8-0

14-0

0-2

99-4

93-3

93-4

99-4

Blood .

Spleen
Liver .
Kidneys
Lungs .
Tiunour
Other tissues
Total urine -
Total faeces .

Becoverv

Selected tissues u-ere then dissected out and assayed for “'Cr. Kesults are sum-
marised in Table I. It can be seen that ^iCT accumulates in both the liver and
the spleen, the highest concentration, but not necessarily the greatest amount
being found in the spleen. In contrast to the observations of Greenfield, Sterling
and Price (1958), uptake in the tumour is negligible or extremely small. The
livers and spleens of animals sacrificed after their haemoljdic episode are found to
be enlarged and engorged ynth blood. It thus appears that the chief sites of red
cell destruction during the episode are the liver and spleen, uiiich together
account for some 40 per cent of the radioactivity lost from the circulating blood.
Pvcd cell destruction due to haemorrhage in the vascular bed of the tumour is
absent or extreme^ small.

Survival of red cells from tumour-bearing donors labelled with ^^Cr and transfused to
normal recipients

August rats receiving tramsfusions of blood from donor animals bearing tumour
Pv 2426 undergo a haemoljdic episode similar to that observed in animals re-
ceiving implants of tumour. Fig. 7-10 show the results of experiments in which
blood was taken from tumour-bearing donors at 4, 9, 14 and 19 days after implan-
tation of tumour and labeUed with ^iCr ; 0-5 ml. of the labelled red cell suspension
or of the supernatant plasma was then transfused intravenou.sly to normal litter
mate recipients. It^en transfusion of red ceUs is carried out at 4 days after
implantation, at which time the growing tumour is scarcely palpable (Fie- 7) the
haeinoljTic episode is preceded by a period of normal survival of the labeUed cells
During the episode, both the transfused cells and the donor’s orvn circulatin<^ red
cells are destroyed simultaneously. Cells transfused at 9 dav« I c

donors own haemohdic epkode (Fi<.. 8) caus7a morf™ ^
a period of normal sunival can stiS be seen. At 14 days {Fig
the recipient is almost immediate and frequently fatSi^ m

234

E. H. EELCHEE AND SHIELEY M, SDIPSON

Jicii tfcinsrusion of colls is ca-rricd mit sif i o -ft. • i

cates tliat the labelled cells m fact have a shortened life span. Nererthehss, it is

Kio. 7, 8, 9, 10. — ^Blood haemoglobin and *>Cr-eoncentration in normal “ August mts
transfused with 0-5 ml. ^iCr-labelled red cells or plasma from litter-mate donors bearing

tumour R 2426.

O Recipients receiving labelled red cell suspension.
X Recipients receiving diluted plasma.

Fig. 7. — ^Blood taken from donor 4 days after implantation.
t / V / I Range of '■'Cr-concentration in normal “ August rats transfused vrith
labelled cells from litter-mate donors.

KBD CELI, DESTSUCTIOS IS TUMOOE BEAEKG EATS

2H5

„,e„ tot to donort cells are not InEotod to any great extent in to haemolytic
episode which destroys the ® a haemolytic episode in the recipient

iStXt &— bnt oUrfng abont A days later

in time.

U..^^ Rnngo of ^'Cr-concentration in normal “August” rats transfused with ^iCr-
Inhcllcd cells from Utter-mate donors.

Pig. 1 1 sliows the results of an experiment comparing the effects of intravenous
and intrapentoneal transfusion of ^’Cr-labelled cells from tumour-bearing donors
to normal recipients. Intrapentoneally injected «Cr-IabeUed ceUs rapidly annear
m the cncuiation and their haemolytic actiiity does not appear to be dwrSsed
by passage through the peritoneal cavity, since no differenermat tfo f t
betivcen the survival of labeUed cells in aifimnlrs tranr i^T ^ observed
that in animals transfused intra^Siur and that

236

E. H. BELCHER AHD SHIRLEY M. SIMPSON

Li furblier experiments, blood Avas taken from a tumour-bearing animal shortly
before it Avas due to undergo its haemolydiic episode and the labelled red cell sus-
pension diluted A\dth isotonic saline. 0-5 ml. portions of the diluted suspension
Avere then transfused intraA^enouslj’’ to normal recipients and the interval between
transfusion and the onset of liaemol 5 ^sis in the recipient measured. Results arc

labelled cells from litter-mate donors,

• -A • rpaWp TT With dilution, the haemolj ic ac transfiisio”

summarised m Table U. ' TYimmired by the mtertml bewe j^-tic

Wood is progressively reduced, ^ of X WO, •

-oSris eor^spouds .0 i e w

mo wy no. . — d W

Table III summarises the results ux

studies.

237

RED

CELL DESTEUCTIOK IK

tumour BEARIKG RATS

“ .iih Tumour R 2420

Material transfused

ISension of red ceUs from non-tumour-bearing donor
Suspension of red cells from tumour-bearmg donor .
Suspension of red cells from tumour-hearing donor .
Sus^nsion of red cells from tumour-bearmg donor .
Suspension of red cells from tumour-bearmg donor .

Dilution

Interval between
transfusion and
onset of

bnemolytic episode
(dny.s)

. Ko episode

• ff

0-1

. 5

0-01

. 7

0-001

Table 111.— Interval Between Transfusion and Onset of Haemolytic Episode m
“ Auqust ” Rats Transfused ivith 0-5 ml. Washed Red Cell Suspen-pon Taken
from Utter-mate Donors at Different Times after Implantation with Tumour

R 2426

Interval between
Interval between transfusion and

implantation and onset of

transfusion haemolytic episode

(days) (days)

4
9

Ko episode
12

3
2

of labelled red cells -was transfused from tumour-bearing donors to normal reci-
pients at different times after implantation of the donors. The haemotytic activity
of the donor’s blood as measured by the rapidity with which it promotes a haemo-
lytic episode increases to a maximum which is reached during or just after tlie
episode in the donor, and then slowly declines. IMiilst the results of experiments
involving different donors are not strictly comparable, an approximate evaluation
of the relative haemolytic activity of the donor’s blood at different times after
implantation may be made by comparison uith the data of Table II.

The relative effects of injections of red cells and of plasma which were reported
in Fig. 7-10 may be similarly evaluated. The results show that the haemohdic
agent is mainly associated Bith the cellular fraction and is only found in the plasma
at a concentration lower by at least two orders of magnitude. ^

The effect of mulliple transfusions of red cells from tumour-bearing donors to normal
recipients

Following the induction of a haemolytic episode in an animal „ +

or a .raosfusio,. from a tamiur-boarmg

to Muco a soaond episode either by tramfusioTor imohn. t T,

.be resoits of ao eaperimeot i„ rvliicb ..Ww'Ss™

238

E. H. BELCHEB AND SHIRLEY M. SIMPSON

recipients from a tumour-bearing litter mate donor during its haemolytic episode
and caused the expected liaemolydiic response. Four weeks later, the recipients
were transfused with ®^Cr-labelled red cells from a second tumour-hearing litter
mate donor also undergoing its haemolytic response. On this occasion, no haemo-
lytic episode is observed, although the same red cell suspension injected into the

„in>aiB previously transfused did not fail to produce the expected p.««, .1

Tult— has been found to

litial haemolytic response. It has undergone its

ransfused from a tumour-bearing , near-normal sun'ival (Fig- '

pisode to a normal recipient haw , jpjent. This is a further exan i

Ithough a haemolytic episode is induced m the recipien

bed cell eestructiok in

of the protection afforded to the red cella of
lytic episode.

tumour bearinc rats

an animal that has nndergonc a haemo

PiQ. 11 . ^Blood haemoglobin and **Cr-concentration in nonnal “August” rats transfused

with 0-5 ml. ‘'Cr-labelled red cells from tumour -bearing litter-mate donor 4 days after
implantation of latter with tumour R 2426.

O Injection by intraperitoneal route.

• Injection by intravenous route.

t / r - 1 Range of ^'Gr.coneentration in nonnal “ August ” rats transfused with ®’Cr-Iabelled
cells from litter-mate donors.

DISCUSSIOK

In the ab.seiice of the results of serological studies which are still incomplete,
detailed dLsoussion of the mechanisms rmderlying the phenomena described must
be speculative. However, it is clear that the haemolytic episode and anaemia
observed after implantation of the tumour R 2426 do not resemble that described
by Greenfield and his co-workers, since no accumulation of ®^Cr in the tumour i.s
observed after transfusion of s'O-labelled red cells.

240

E. H. BELCHER AND SHIRLEY M. SIJXPSON

Fig. 12a.

Fio. 12. — Blood hnemoglobin and ®‘Cr-concentration in normal “ August ” rats transfused
on two occasions at an interval of 4 weeks with 0-5 ml. ®‘Cr-!abelled red cells from tumour-
bearing litter -mate donors 10 days after implantation of latter with tumour R 2426.

A Response to first transfusion.

B Response to second transfusion.

1/ / ^ Range of ®’Cr-concentration in normal “ August ” rats transfused with *'Cr-]abcIIc<I
cells from litter-mate donors.

tion of on the liver and spleen during the episode. Such agglutinins
be evoked in response to the tumour graft in a manner similar to that in «
antibodies to tumoim or skin homografts which may be titrated as "

tinins are produced in mice (Hildeman and Medawar, IfloO). Alternatnc j, >.
might be produced by the tumoiu’ itself against the host animal’s cells as suggc-

beb cebb BESTEBCTIOB- ns- tbmobb BEAE.NG e^ts

241

1 Tn either of these situations, the

bv Green, Wakefield and receiving a transfusion of bloocl from

Sluction of haemolysis in

a tumour-hearing donor rvould be due to tM demonstrated that a

it appears improbable that sufficient antibody could be transferred by such a
mechanism to cause the haemolysis of nearly all the host’s cells.

A further possibility is that the tumour might produce an antigen similar or
complementary to antigenic components on the host’s red cells. Antibodies pro-
duced by the host against this antigen might then induce agglutination of the red
cells. The antigen could be transferred with the blood of a tumour-bearing donor
to a normal rccipietit and induce a similar production of antibody and agglutination
of the rceijhent’s red cells. However, the finding that the time between transfusion

240

E. -H. SELCHBR ANV SHIRLEY M. SIMPSOK

a «WoS i„

aggtottains (Dade. 1954 Jandl, Jo«a .„d

Fio. I2a.

Fig. 13. — Blood haemoglobin and =‘Cr-concentrntion in normal “ August ” rnfs trnnsfiisocl
on txro occasions at an interval of 4 weeks with 0-5 ml. ^iCr-lnbellcd red cells from lutnour-
bearing litter-mate donors 10 days after implantation of latter with tumour li 14211
A Response to first transfusion.

B Response to second transfusion.

\7^'7~71 Range of =>Cr-coneentration in normal “ August ” rat.s tran.sfiisod with ‘'Cr-Inbellnl
cells from litter-mate donors.

tion of on the liver and spleen during the episode. Siicli agghitmins mip i
be evoked in response to the tumour graft in a manner similar to that in nine .
antibodies to tumour or skin homografts rvlnch may 'J hev

tinins are produced in mice (Hildeman and Medawar, lOofl).
might be produced by the tumour itself against the host animal .s cell.s as .sii^.,t.

241

bed ceee destbuctioe en tumour BEARINO rats

1 /ia:;Q\ Tn either of these situations, the
by Green, Wakefield and I receiving a transfusion of hlood from

induction of haemolysis in ^ normal am antibody from the donor s

a tumouT'bearmg donor However it has been demonstrated that ji

il apiioars improbable that sufficient antibody could be transferred by such a
mechanism to cause the haeniol3"sis of nearly all the host’s cells.

further jiossibility is that the tumour might produce an antigen similar or
comp cmentary to antigenic components on the host’s red cells. Antibodies pro-

242

E. H. BELCHER AND SHIRLEY JI. SIMPSON

and onset of the haemolytic episode may be as little as two days does not seem to
be compatible Math such a mechanism.

The obsen’-ations that the haemolytic activity of the blood increases steadilv
until time after implantation of tumour and that a haemolytic episode can follow
the transfusion of mmute amounts of blood from a tumour-bearing donor to a
normal recipient suggest that the responsible agent is some factor, possibly viral
in nature, which is capable of multiplication -within the host. If such is the case
the (pestion must be asked whether the Yirus is the causative agent of the tumour
or whether it exists concomitantly in the tumour tissue but without aetiolorical
relationship to it (Zilber, 1958). Further studies designed to test all of these possi-
bilities are being carried out.

SUMMARY

Rats of the “ August ” strain hearing the transplantable adenocarcinoma
R 2426 undergo an acute liaemolytic episode 10-15 days after implantation of
tumour. This phenomenon has been investigated by radioactive tracer techniques
with and ^®Fe. Cells are destroyed randoml}'^ ndthout regard to age during
the haemolytic episode. Destroyed red cells accumulate primarily in the liver
and spleen, destruction in the vascular bed of the tumour being absent or extremely
small.

A similar haemolytic episode may be induced in normal animals transfused
intravenously or intraperitoneally, with blood from tumour-bearing Jitter-mate
donors. The rapidity of onset of the episode is related to the amount of blood
transfused and to the time after implantation at which the blood is taken from
the donor.

After an initial haemolytic episode induced by implant or transfusion, it is
impossible to induce a second episode in the same animal ; furthermore, red cells
from a tumour-bearing donor which has suffered a haemolytic episode show near-
normal survival when transfused to normal recipients, although they provoke a
haemolytic episode in the recipient.

The mechanisms by which these phenomena are brought about are discussed.

This ivork has been carried out in part at the Institute of Cancer Research.
Royml Cancer Hospital, and in part at the Postgraduate Medical School, and has
latterly been supported by a grant from tlie British Empire Cancer Campaign.
The technical assistance of Mr. H. C. Christie, 3Iiss Frances Holmes and Jhss Judit i
Topper is acknowledged.

REFERENCES

Belcher E H — (1958) 3rd International Symposium on Radioactive

Medioine '.„d Eese.rch, B.d G.M™. Munich (Crton »nd Schwm...

berel v. 206.— (1959) Jefa I7n. ini. Ca/icr, 15, Sdb.

Idem AND Haeriss, E. B.— (1957) J. 4 s’dO.

Idem, Lamekton, L. F. and Habbiss, E. B. (1^®) ^ J- ' } 44 , sCO.

Beblin, N. L, Lawbence, J. H. a™ Lee H. C--(I954) .7. cOn. Aicu„

BeeliN, R.— (1951) Ada med. scand. n9, Suppl. 25-, P- i- Acquired ’. lAmdon

Dacies, j. V.— (1954) ‘ The Haemolytic Anaemias, Congenital ana Acquirt

(Churohill).

RED CELL DESTRUCTION IN TUJIOUR REARING RATS 2411

Ekrekstein, G. — (1958) Ada physiol, scand., 44, 80.

Green, H. N., Wakefield, June ..vnd Littlerwood, G. — (19.57) Bril. incd. J., ii, 770.
Greenfield, R. E., Godfrey, J. E. and Price, V. E. — (1058) J. val. Cancer JvsI., 21,
641.

Idem, Sterling, W, R, and Price, V. E. — (10.58) Ibid., 21, 1000.

Hildeilan, W. H. and Medaivar, P. B. — (1059) Immunoloyy, 2. 44.

Hitman, G. A., Gellhorn, A. and Harvey, J. L. — (1050) Blood, 9, 018.

Jandl, j. H., Jones, A. R. and Castle, AV. B. — (1957) J. din. Invest., 36, 1428.
jMiller, a., Chodos, R. B., Emerson, C. P. and Ross, J. E. — (1050) Ibid., 35, 1248.
Price, V. E. and Greenfield, R. E. — (1058) Advanc. Cancer lies., 5, 190.
lidem. Sterling, W. R. and jMacCardle, R. C.— (1950) J. not. Cancer Ins!., 22, 877.
Ultmann, j. E., Fish, AV. and Hyman, G. A.— (1050) Cancer Res., 16, 885.

ZiLBER, L. A. — (1956) Advanc. Cancer Res., 5, 291.

244

STUDIES ON" MOUSE LEUKAEMIA. THE FATE OF

HOMOGEAFTS IN imm^OLOGICALLY TOLEEANT MI®

J. F. A. P. mLLER

From the Chester Beatty Research Institute, Institute of Cancer Research :
Royal Cancer Hospital, Fulham Road, London,

Beceived for publication March 28, I960

Noejiai. tissues have been successful]}’' transplanted in foreign liosts which
h&\ e been treated in such a ivay as to render them tolerant. Such tissues incliule
skin (Billingham, Brent and Medarvar, 1953), th}Toid (Woodruff and Boswell,
1954 ; Woodruff and Sparrow^, 1958), ovaries (ICrohn, 1958 ; Martinez, Smith and
Good, 1958), adrenals (Medaivar and Bussell, 1958), and pituitaries (Martinez
el al., 1958). The homotransplanted glands retain their functional integrih' in
the immunologically tolerant hosts : thyroid homografts are able to concentrate
radio-iodine, adrenal homografts can sustain life in adrenalectomized animals
on a salt-deficient diet, ovarian homografts give rise to sexual cycles and, when
orthotopically transplanted, can produce ova from which litters eventually deve-
lop, and pituitary homografts can maintain normal groudh and norma! control
over other endocrine glands.

Acquired tolerance of homografts of normal thymuses has been achieved in
the experiments reported here by the intravenous inoculation during the neonatal
period of living spleen or thymus cells taken from adult mice of the same strain
as the prospective donors of the thymuses. It is now well established that lymplio-
cytic leukaemia, whether spontaneous or induced, does not usually develop in
the absence of thymus tissue (McEndy, Boon and Furth, 1944 ; Kaplan, I flat);
Law and Miller, 1950a, b; Gross, 1959; Levinthal, Buffett and Furth, 19.19.
IMiller, 1959a, 6 ; 1960b) and that thymus grafting restores the potentiality for
leukaemia development in isologous combinations (Law and Jliller, lOSOn, v.
Kaplan and BrovTi, 1954 ; Miller, 19596, 19606). It will be shown in this papr
that thymus tissue from genetically susceptible mice can undergo niaiignan
transformation in a foreign but tolerant host, and that a number of such nialign.w
thymuses can be made to regress completely following the inoculation of actna f<
immunologically competent cells.

3IATEKIALS AND JIETHODS

Mice.— Mce of the C3H/PW or C3Hf/PW strain, inbred in our
since their acquisition from Bittner in 1938, show an incidence o ‘
iyunphocytic leukaemia low’er than 5 per cent after 15 montl^ of age, *.p
eous tumours of other tj’pes are also very rare, except in G3H fema e in
80 per cent of which usually develop mammary tumours after 8 montiis.

The Akf strain, originally obtained from Dr. J. Furth via L Ap" of

breth-HoIm, has been inbred here since 1945 and shows a lug i i

THYJIUS HOMOGRAFTS IX

V IMMUXOLOGICALLV TOLKRANT MICK

245

cent of the mice developing

spontaneous Ijnnphocytic leukaemia, loL)

the disease at approximatelj 9 moi susnensions from tlivmiis or spleen

out, U .0 orgl, u iu

tfeerStaSr l*ospiL Bolutiou. "asWog twice ami '".,1 1

loe'ml. of the suepeusion — ' ^^AVlui c

sTrs irir^cs“"^;e i“X^c”Suf ...e.,

is Imerior fS vein or the sigmoid sinus of the new-born mouse hn_s hcen
described and illustrated in the papers of Billingham and Brent (IhoO, IPoO) am

in a recent article b 5 " Brent (1959). m- i

l^Iarrow was expelled from the shafts of the femurs with a jet of Binger Jilios-
phate tlirough a No. 14 gauge needle mounted on a sjTinge. Gentle agitation by
suction in and out of a pipette allowed the cells to separate from one another.
They were then washed tivice and resuspended so that each new-born mouse
received about 8 million cells.

Thymectomy and thymus grafting.— Thymectomy was performed at 3 to n
weeks of age as described previously (^.liller, \960{>). Each thymectomixed mouse
was given a subcutaneous graft of a whole thymus from an untreated new-born
C3H or Ak female mouse, as required, on the day of thjTnectomy. Thymuse.s
from new-born mice were removed aseptically and introduced by a sterile trocar
into the subcutaneou.s tissues of the right (C3H thjnnusesJ or left (Ak thymuses)
axilla. The mice were thereupon given 3000 to 4000 units of penicillin and 3 to
0 mg. of streptomycin daily for about 10 days to guard against infection.

Skin grafts . — Skin grafts from 1- to 2-month-old female Ak or C3H mice were
transplanted to 6- to 8-week-old C3H or Ak mice by the method of Billingham
and lledawar (1951) to provide an external indicator of the tolerant state.

Adoptive immunization . — ^The immune state may be acquired in three ways ;
(1) actively, by the introduction of an antigen into the animal, (2) passively,
by tlie introduction of antibody prepared in another animal, and (3) by the
transfer of immunologically activated cells from one animal to the other.
Billingliam, Brent and Medawar (1954) have named the state of immunity
acquired in thi.s third way “ adoptive ” immunity.

Normal 2-month-old C3H female mice were immunized against normal Ak
ti.ssue.s. Eacli C3H mouse was given bilateral skin grafts and an intraperitoneal
injection of cells from two thjnnuses and two spleens from 1- to 2-month-old
lieaUhy Ak donors. Ten to eleven days later, at a time when the reaction in the
slun graft was most intense, the mice were killed, and cell suspensions were
prepared from their axillary^ and inguinal lymph nodes and spleen The cells
were mime(hately injected intraperitoneally into C3H mice tolerant of Ak to
abi ogat e tolerance. Each tolerant host received cells from two spleens and tirelve

I assage A filtrate . — ^This most powerful leukaemoeenic filtrate -n-ao i

roin leiikaeniic mice of the G3Hf/Gs strain as described previously

246

J. F. A. P, MILLER

million cells Ting was injected intraperitoneally into untreated adult Cffl aur)

Ak mice. Small pieces of leukaemic thymus were introduced as tic Uv n
trocar under the skin m some of the mice. asepticalh bj

Histology.— Sections were fixed in Bouin’s fluid and stained in haeraafosvlin
and eosin or other stams when indicated.

EXPERUIEXTAL

Experiments were performed on both Ak mice tolerant of CSH and on C3H
mice tolerant of Ak. The former were divided into tlmee groups accoiding f o the
1 injected intravenously at birth. Group 1 received C3H or

G3Hf thymus cells, group 2, C3H spleen cells and group 3, C3H marrow cells.
Some of the mice in group 1 also received an injection of Passage A filtrate,
usually 3 to 5 da 3 ^s after birth. Wien inoculated as late as 14 da 3 's after birtl’i
(Miller, 1960a) this filtrate causes leukaemia to develop within 3 to 6 montlis in
100 per cent of non-thyniectomized mice of the Ale strain (Jliller, lOOOa). All
the mice were th 3 ’'mectomized at 3 to 5 rveeks of age. In group 1, they were grafted
subcutaneous^ with a da 3 ''-old C3H or C3Hf th 3 Tnus. In groups 2 and 3 tliei'
received isologous tlijmus grafts.

C3H mice made tolerant of Ak were divided into two groups. Jlice in both
groups were th 3 Tnectomized but those in group 1 were grafted with day-old Ak
thy'muses while those in group 2 were grafted with da 3 ’^-old C3H th 3 ’muses.

BXPLANTATION OF PLATES

Fig. 1. — ^Jfonnal subcutaneous C3H tajinus graft in an Ak mouse tolerant of CSH. Tiiis
section was made 60 days after grafting.

Fig. 2 . — Jlammary adenocarcinoma in an Ak female mouse which received CSH marrow
cells at birth.

Fig. 3. — Slalignancy in a subcutaneous Ak thjTnus grafted to a tolerant CSH mouse.

Fig. 4. — High power view of leukaemic Ij-mphocytes in thjTnus graft seen in Fig. 3. Rote
numerous mitoses.

Fig. 5. — ^Honnal salivarj- gland tissue separated by a thin connective tissue capsule from the
parotid gland tumour. Note the resemblance between the normal salivarj- ducts and tlic
duct-like elements of the tumour.

Fig. 6. — ^Another salivary gland tumour showing clearly the duct-like pattern plus the loow
mesenchjrmal elements.

Pro. 7 , — more solid tj^pe of salivarj^ gland tumour. the adenomatous pattern merging

into a confluent mass of cells.

Fig . S. — High power view of a salivar 3 ' gland tumour. Numerous mitotic figures arc seen. a
cells still show some grouping into glandular elements.

Pig. 9. — A. pleomorphic sarcoma. Note the tumour giant yells and undifferentiate pattern
of this tumour. ...

Fig. 10. — ^High power view of sarcoma seen in Fig. 9 to emphasize the nuclear \arintion an
numerous mitoses.

Fig. 11. — fibrosarcoma infiltrating skeletal muscle.

Fig. 12.— a tumour from the upper eyelid. Note the well-differentiated pattern and huntfles

of uniform cells. . i tl /.

Fig. 13. — necrotic anaplastic carcinoma. The grouping of the cells into clumps an i
degenerative changes are clearly shown. i i n.

Fig. 14. — kidney from a mouse ivith bilateral parotid tumours. >ote the irrcgti ar imp i
c\-tio infiltration in the cortex.

Fig. 15.— High power view of kidney shoivn in Fig. 14. Note the normal .small Ijmphocite
grouped around blood vessels and the absence of mitoses.

Bp.ITISir.JoUltNAI, or Ca>-CKR.

Xo.

MiJler.

THYMUS HOMOGRAFTS IFT IMMUNOUOGICALLY TOLERANT ^IICE

247

RESULTS

The results are presented in Tables I to V.

Induction of immunological tolerance in CHH or Ak mice

Tolerance of Ak skin gi-afts in CSH mice injected at birth with Ak spleen or
thymus cells has already been described (Miller, IDGOfir) and the residts are re-
peated here for comparison with tolerance of C3H skin grafts in the Ak strain.
Up to 90 per cent of C3H mice were tolerant for periods up to a year or more.
There was no evidence that spleen cells were more effective than thymns cells
in inducing tolerance as they airpeared to be in the strain combination used by
Billingham and Brent (1959), Injection of C3H spleen, thymus or marro\v cells
in Ak new-born mice induced tolerance of C3H skin grafts in 70 to 80 per cent
of the mice (Table I). The slcin gi'afts were intact for a period of 4 to 6 months,

Table I. — Tolerance of Skin Grafts in Mice Inoculated at Birth with 5 to 8 million
Spleen, Thymns or Marrow Cells

Strain of I.V.I. given at birth Number

recipient r — '' v m Number of mice

mice Donor strain Cell tr-po group fully tolerant

143 . 128 (80%)*

101 . 13- (8.0%)*

57 . 0 (0%)t

75 . 51 (08%)t

27 . 18 (C7%)t

32 . 20 (82%)f

30 . 0 ( 0%)§

* Ak skin graft intact for a total period of observation of 12 to 18 months,
t Ak skin graft rejected in 10 to 12 days,
s ooS intact for 4 to 0 months (see text),

s C3H skin graft rejected in 9 to 11 days.

C3H/P\V fAk, /Spleen

or I \Tlmmus .

C3Hf/PW LNo cells ‘ — .

("CSH/PW r Thymus .

Ak( j or < Spleen

i C3Hf/PW fMarrow .

pNo cells —

iut thereafter the hair in some of the grafts began to fade in colour and diminish
10 thickness leaving a greyish-white patch. There was, however, no reaction such
fs IS characteristically in skin grafted to non-tolerant mice. Ten Ak mice not
ihcluded in Table I, inoculated at birth Amth C3H thymus cells, were runts and
‘lied before 5 weeks of age.

Tolerance of thymus homografts was evident from sections made of such
gra ts 30 or more days after grafting (Fig. 1). Such thymuses showed intact
orpliology. Thymus grafts in tolerant mice could often be made out as small
1 Jcutaneou.s nodules in or under the axilla when the overlying skin was stretched.

The development of lymphocytic leukaemia and other tumours in Ak mice tolerant

withV^SH If Table II that only one thymectomized Ak mouse grafted

bm narat ^ developed leukaemia. At autopsy the lymph nodes, including
"hiltrated™'^ fi involved and the liver and spleen were extensively

cutan'oouq’rwTf evidence of incomplete thynnectomy. The sub-

haemiu areu ;“y"ins graft could not be found. On transplantation, the leu-

ii oriuinaSV" “’I® I” non-tolerant C3H mice, suggesting that

g nated from lymphoid cells of the thymectomized Ak host.

1 0

248

J- F. A. P. MILLER,

Jm^ufTf;^z7pZ:Tipi ^s'iz'aZ &iS

or CZHfjPW Mice*

I.V.I. cells
nfc birth

Groupt

Type

Strain

rC3H

Thymus

lc3Hf

Spleen

C3H

Marrow

C3H

ITumber

{

5
II

6
11

Mice u-,th mammarj- Mice mtl, hmnhocrtic
^^ours neoplasms '

' ^ — >

Sex

umber

Age in
months

/ —

Number

Age in
monllu

6-13

(average 9)

—

. —

—

12, 14

7-9{

(nveragoS)

—

CLir.

(average 9)

9,10

6-Sj

(average 7)

—

5-l«

(nvenigc S)

* Total period of observation 18 months.

t groups were tlijuneotomized. Those in group 1 were grafted subcutnnrously «itli

LdH or C3Hf daj'-old thjTnuses. Those in groups 2 and 3 were grafted subcutaneously "'itii bologoin
thymuses and received 3 further injections of spleen and marrow cells, respectivclv, at 4. fi nnl 8
weeks of ago.

J These mice received in addition to C3Hf thymus cells, an injection of Pns.snco A filtrate 3 to ■’>
days after birth.

None of the other mice in group 1 developed leukaemia or lymphoid tumour
in the thj'-mus graft, not even those inoculated with Passage A. Oit the oilier
hand, half the female mice injected at birth noth normal C3H (but not C.lHf)
thjTnus cells developed mammary carinomas (Pig. 2). Onlj' one such tumour
has been seen in over a hundred non-tolerant Ak female mice tlie life of wliicli iuul
been prolonged hy tlijonectomy. None has ever been seen in intact Ak femali'
mice of our colony,

(Mice in groups 2 and 3 which were tliymectomized and grafted with isologom
thymuses developed lymphocytic leukaemia at approximately tiie age and fre-
quency characteristic of the AJe strain. In most cases the snbciitancou.'! '
thymus graft was involved. Repeated injections of C3H spleen or marrou co.-
failed to alter the incidence. Some of the female mice in these two groups a
developed mammary carcinomas.

Incidence of lymphoid and other tumours in C3H mice tolerant of Ah

The incidence of tumours in thymectomized C3H mice tolerant of Ak a”
bearing Ale or C3H thymus grafts is shoum in Table III. . , ,

In group 1 (C3H mice bearing AJe thymus grafts) 14 mice ^ L),[.

cytic neoplasms. In 10 the first sign was progressive enlargement at ic ^ ,

subcutaneous thymus graft. In the other 4, generalized lymp i no< ®
ment -was evident from the onset and the graft was involved 3) J j, [,^11
process in only two. One C3H mouse, not included in Table 1 ) * iii,, ^vliich

rejected an Ak skin graft on two occasions, developed a tumour in le < .
later proved to be a lymphoid tumour.

240

.hy®s eomogkatts « Brnm-mooicALEY toeekant ,nc«

JUce with Ijuiphomas

Mico witli otlicr tMmnurx

Groupt

r 29 T n\

rC3H

Aki

C3H

C3Hf

^C3Hf 1^22

total 9"

fC3H

C3Hf

total

(average 9)
5-8

(average a-O)
9,10
10

(5-7)

(0-8)

(7-12)

SOT. KA
SOT, CA

>1.T.

♦ Total period ^ birth with normal Ak spleen or thi-mus cells, thy.noetomi/.ecl

. jixs?d.:”s s ihA- Mi» i" ir»i' * ""

they were grafted with a day-old C3H thjTnus.

M.T. = mammarj' tnmours.

SGT = salivary gland tumours 1 ^ .

SA = sarcoma V described m text.

CA = carcinoma

The histological appearance of a tj^pical Ijonplioid tumour arising in a thjanus

graft is showTi in Fig 3 and 4. . ... ,v i a

None of the mice in group 2 (tolerant C3H mice hearing C3H thymus grafts)

developed lymphocytic neoplasms.

The C3H females in both groups developed mammary carcinomas characteristic
of the C3H strain. Eleven mice in group 1, however, unexpectedty developed a
whole array of tumours which are described below. It is significant that these
eleven mice were survivors from two litters injected on the same day until the
same ]ircparation of a mixture of normal Ak spleen and thymus cells, and that
they were not inoculated with Passage A or other leukaemogenic filtrate.

Immvnorjenetic behaviour of lymphoid tumours arising in 03H mice tolerant of Ak

Tlic lymphoid tumours developing in tolerant C3H mice were transplanted to
both Ak mice and non-tolerant C3H adult mice (Table IV). Five were trans-
]ilantahlc to both Ak and C3H, two grew only in Ak, and five, curiously enough,
only in C3H. Four of the latter had originated when their host was 10 months
old or older and one at 6 months of age, after the host had spontaneously reiected
the Ak skin graft. ■’

Fak of lymphoid tumours in thymus homografts following adoptive immunization
Malignancy in the subcutaneous thymus homografts became evident with

u'.acheil half a centimetre in the largest d^amSo^ Tb ! ^

i,. , W Ot .„c „,oe. This » WedVr^S

230

J- T’. A. P. JIILLEK

Number

6
7
S
0

J5J

Sex

Age in
months at
sacrifice
12
16
5
9
9
5
10
8

Results of trnnsplniitntion In- cells

In strain of origin

In Ak straiiv

* Numerator

Result*

4/6
3/6
0/6
4/4
3/3
0/4
3/5
3/3
3/4
315
2li
4/4

: number of takes ; denominator

Latencyf (days)
21-40
41-48

30- 38
34-38

31- 47
27-31
36-47
20-31
31,33
25-39

Result

0/6

6/0

4/4

2/6

4/4

0/4

3/3

0/3

4/4

3/4

0/4

Latency (days)

20-3(1

24-3(i

36.41

18-30

20-2, I

art_o((

26-32

; number of animaU.

t Interval between Rafting and sacrifice or death.

J This mouse is not included in Table III because its tumour arose after it had rejcrfcd the Ak
skin graft.

^ Tiie first evidence of malignancy in mice Nos. 3, 4, 5, 6, S, 10, 14 and 15 was enlargement at (lie
site of the thymus graft.

Table V. — Effect of Immune C3H Cells on Li/mphoid Tumour Orowth in C3U

Mice Tolerant of Ak

Tran.splantntioii

Age at Interval in daj's between results (Table JV)

onset of , * , , ,

Number

malignancy'

diagnosis* and

diagnosis

Takes

Tako^

Sex

(months)

1st treatment

and death

in xVk

in C3H

4 -

■f

Not done

-f

-r

* The da3' of diagnosis was arbitrarily li.ved as the day when the enlargement of (he graft liatl
reached half a centimetre in its largest diameter.

f Tumour in thymus graft completely^ regressed from 20 to 40 days after first treatment niili
immune cells.

reaction in the skin graft during the second tveek after tlie first injection of innoom
cells. No signs of generalized leukaemia were ever present in tliese tlirco mice.

The otlier four mice all succumbed to tJie disease. A feeble .skin gr.nft renettoo
was observed in only two and signs of dissemination of the disease became e4 1 ( p!>|
during the first or second week of treatment. The mice ncro Ivilicd v ten i
became obvious that the treatment had failed, and the tumours were trans-
planted. As seen from Table IV, only one of tliesc^ tumours took only m Ak.
The other three were transplantable to both Ak and C3H.

The development of parotid and other hmonrs in C3H mice inoculated at hirlh mth
normal Ak spleen and thymus cells

The most unexpected result of tlie present experiments nas flie occntTfrice o

parotid and sublingual salivarj' tumours,

intramuscular .sarcoma.s and olbcr

THYilUS HOMOGRAFTS IM IMMUNOLOGICALLV TOHEIIAXT .MI(!E

2:11

tumours in eleven C3Hf mice from two litters inoculated at birlli with (he same
Busuension of normal Air spleen and thjnnus cells (Fig. 5-1.1). Mo.sl. of the mice
had salivarj- gland tumours on both sides (cither both jiarotuls or one ])aro(.ul
and one sublingual gland being involved). One mouse had three ]irimary tnmom .s
(one parotid gland tumour, one sarcoma in the pectoral muscles and one saicoma
in the thigh muscles).

All the parotid tumours examined showed essentially the same stnicf.iirc.
ilacroscopically they were made up of discrete nodules, (inn, opaque, yellowish-
white and of rubber}' consistency. Alicroscojiically, a thin (ibrous capsule^ could
he seen in some places only with the growth c.xpanding and comjiressing it.
Outside the capsule was some lymphocytic infiltration. Two main types of
structure were found in the tumour proper : duct-like structures very similar
to the normal ducts of the gland (Fig. 5) and a uniform pojmlation of mcsenchymal-
like cells with oval nuclei and delicate cjiioplasmic processes. Fig. (i shows both
these patterns while Fig. 7 shows a more solid growth in which the adenomatous
pattern merges into a confluent mass of cells. Under the high jrower (Fig. S)

many mitoses were seen in both the duct and mesenchymal cells. There were no
necrotic or degenerative changes in the tumour as a whole.

Several mice had sarcomatous tumours growing in muscle tissue. These
tumours were whitish-pink solid circumscribed masses without necrosis. The
istological features of two such tumours are shown in Fig. 0-11. One of the
umoiTOwasin the pectoral muscles and showed much individual celhdar varia-
mn (Fig. g and 10), Alost of the cells were large with big nuclei and a greatly
^uclsar-cytoplasmic ratio. Tumour giant cells were plentiful and
Cell boundaries were hard to see in some areas and a diffuse
muO present. Many new blood spaces could be seen and invasion of

"hen evident. No cross-striation could be found in the tumour cells

tumour stained with phosphotungstic acid haematoxylin and the

"arcom'^ best described as un undifferentiated anaplastic sarcoma. Another
"We mVn r mouse was growdng in the muscles of the thigh. The eells
giant cell nuelei Avere oval and only an occasional

"asrichlv"^^ growth could be seen invading skeletal muscle and

individual c^ii^ ’ bhin-rvalled, blood vessels. Fine fibrils ran between

n consideraW * special stains showed that the tumour cells were producing
^ne Tuous^ ®^°unt of collagen. The tumour was imdoubtedly a fibrosarcoma,
n.vclids (Fig n bilateral parotid tumour and tumours growing in both upper
vcntinictre in d~ latter w'ere excised when they reached about half a

jngically, they nnd appeared to consist mostly of fibrous tissue. BUsto-

\vcje V ''^.'"eh-differentiated groiHhs underlying normal epidermis.
I'nttcm with somp V ^ bundles of uniform cells running in an interlacing
f ndcr the hiffh wowa nuclei although this was not w'ell marked.

hcnimT npiirrxfii " nuclear variation than would be expected

■’'grating the histowTr mitotic figures w'ere present,

mouse had a ‘^'^gnosis of fibrosarcoma of low'-grade malignancy.

110 ^' find refl^r!^ powdng in the abdominal cavity. It was a spherical

, I'l w i)o 1 p patches of necrosis and h aftmnrrTiQfTA 4.^ 4-1,^

lof 'r'e of tlie left kidnp? « f t necrosis and haemorrhage, attached to the
.. '"''■cnal could not Im bhe kidney itself. The

‘'''^•''ngedincluin™ ;T ^^differentiated cells

ps and showTng numerous mitoses. Invasion of hinnd

numerous mitoses. Invasion of blood

252

J- P. A. P. MILPEE

card77ma frequent. The tumour was diagnosed as an undifferentiated anaplasi

nc if! proliferative or nuclear changes were observed in the renal tubules sik
as has been described m mice inoculated with the polyoma virus (Stewart Fdc'
and Staton, 1959 ; Staton e< ol.. 1959). ThereU an irregUr 1™*“
nffltration in the “rtex of one kidney, many small round cells being gtomi
mostly around small blood vessels. No inflammation was seen in the renal tubul
nor was there any other evidence of pyelonephritis. The cells, themsclvi
appeared to be normal smaU lymphocytes (Fig. 14, 15) and no mitoses were fouii
IJiere was no evidence of any leukaemic process anj^vdiere in the animal.

Much of the above histological description was verjf kindly supplied by 1
P. M. Sutton to ■\vhom I am ver 3 '' grateful.

BISCUSSION

It has been observed in the experiments reported here that high-leuknen
strain Ak mice which had been made immunologically tolerant of C3H tissi
and which had heen thymectomized and grafted voth C3H thjmius tissue did )
develop Ijmaphocytic leukaemia or lymphoid tumours in the graft. These in
are said to contain a leukaemogenic agent (Gross, 1958) and some of them reccivi
soon after birth, an injection of Passage A filtrate, which has been shown to acc(
rate the onset of the disease in intact Ak mice (Mfller, 1960a). Yet, in spite
the presence in a genetically predisposed strain of both tlijunus tissue (gcnctic/i
foreign but tolerated) and leukaemic agent, the disease failed to develop. <
the other hand, low-leukaemic thymectomized C3H mice tolerant of Ak and be.
ing subcutaneous grafts of normal new-born Ak tly^mus developed Jymiihocy
leukaemia or malignancy in the graft as earlj’^ as 5 months after birth. The mi
received no injection of leukaemogem’c filtrate at birth although thej' were inject
with normal Ak cells. However, we have failed to demonstrate that such i
injection of cells, per se, produced leukaemia in tolerant thymectomized C3H mi
bearing isologous thj'^mus grafts (Table II) or in tolerant C3H mice with intc
thymuses (Miller, 1960a). Clearly, therefore, the genetic susceptibility to Ic-
kaemia development must depend on an intrinsic property of the thymus ti.'.si
itself. The results obtained here are in accordance with those of Law ( 1 952, i 95 ■
who showed that “ genetically tolerant ” Fj hybrids from crosses between liij;
and low leukaemic strains, bearing tlijunus grafts from the low leukaemic parciif:
line, did not show neoplastic change in the grafted thymus, whereas tliose rcc(i\
ing thvmius grafts from the high leukaemic parental line developed a high mcHlcni
of lymphocvdiic neoplasms in the graft. In similar experiments, vap an, Wb
and Brown (1956) showed that C57B1, but not CSH. thjmne implants develop,
lymphoid tumours in irradiated thymectomized (Co/Bl X L3H)b, tiosts

There are clearly two possible ways by wbicb the presence of Ak hj m
in a tolerant C3H host could lead to the occurrence °/^Ampho^Lc •

the host. Either (1) the Ak thymus is a source of „ ,1^ oi

hTnphoc 3 Tes which can undergo neoplastic transforma ion n <

host or in a genetically foreign but tolerant host ; or 2) a non-cellular mfluu

from the Ak thjmius is responsible for ^’'.^.'J^kfi^Tmpbocvti^ neoj.ln^

It would be expected on the first hjiiothesis that the h nipf.oc^ i

THYMUS HOJIOGRAHTS IN IMMUNOLOGICALUY TOU15HANT MlOh

2r)H

.shich developed in tolerant C3H mice would bo transplantable to Ak mice.
Two of the early neoplasms were undoubtedly of Ak origin, growing only in Ak
mice, and those that regressed following injection of sensitized lyiniih node and
spleen cells must presumably also have been of Ak origin. Ihc behaviour ol the
5 neoplasms which grew both in Ak and C3H could be explained by reference to
previous work which showed that a certain percentage of Ak leukaemias would
takeinCSH (Furth, Boon and Kaliss, 1944) or that, by transformation or iinmuno-
seiection or both, a single passage of a tumour through a tolerant foreign host
allowed subsequent growth in untreated adult mice of the foreign strain (Kojnow-
ski, Gail and Love, 1956). Finally, the five neoplasms that grew only in C:iH could
be spontaneous C3H neoplasms that would have devclojied wliether tlie Ale
thymus was present or not. At least the one arising at 1 6 months is likely to have
been a spontaneous neoplasm. The disease is, however, rare before 12 months
of age and only 2 cases have been diagnosed at 12 and 14 months in 227 untreated
mice obsen'ed for a period of 14 months (Miller, 1960a).

The second hypothesis assumes that a malignant change takes place in a
population of lymphocytes, host or donor, as a result of a non-cellular influence
from the thymic epithelial reticulum cells of the Ak donor. The lymphocydic
population of the compatible graft (Air) in the genetically different but tolerant
host (C3H) might undergo a change, donor-type lymphocytes being gradually
replaced by host lymphocytes. On the other hand, the retieular tissue of the
donor thymus might survive and induce neoplastic transformation in either
donor or infiltrating host lymphocytes. This may account for the fact that most
of the early neoplasms were Ak in type and the later ones C3H. Again, this
situation is similar to that described by Law (1952) and Law and Potter (1956).
m their experiments, AKR thymus fragments increased the incidence of leu-
kaemia in (03Hb x AKR)F]^ hosts, the resulting neoplasm being transplantable
only to Fj hosts. In another hybrid combination, susceptible to the leukaemo-
gonic activity of X-irradiation, malignancy developed in thymuses from unir-
'■a lated C57B1 donors grafted to irradiated (C57B1 x A)Fi hosts. The tumours
oping early (at about 5 months) were found to be contributed by descendants
onor C57B1 thymus tissue whereas those arising later (7-10 months) were
oun to have originated from Fj host cells which must have populated the graft,
ours of immunity against homografts of skin and transplantable tum-

'iiitl ^ode cells of actively immunized mice has been described by many

iPofter, Taylor and MacDowell, 1938 ; MacDowell et al, 1938 ; Brncic
Gasic, 1952 ; Mitchison, 1953, 1954 ; Billingham, Brent and
1956 ; Koprowski et al., 1956). By using activated lymphoid
making RussellJ1958) showed that tolerant adrenalectomized mice

Hoecker and
medawar, 1954
Medawi

miwd ’a,ui . of adrenal cortical tissue could in effect be adrenalecto-
^'Pmn) witb3n7®®“ “ obtained by Krolm (1960, personal communi-

'''^'‘^dbed hero r w homografts of ovaries In three out of seven experiments
h'll.v causedTmm,fn5r-^ n ."f t ^ success-

hivmus aC no f ®hin, and
Ejected wt traS fhat failed to

too advancenSrt f I fhe disease

h'^t could not be f commenced. The other three tumours

•■"'y immunitv S ^ acquired the ability to overcome

«>tv of adoptive origin in the tolerant hosts for on tLsplISS

254

J. F. A. P. MILLER

tiiey grew perfectly well in both non-tolerant adult C3H hosts and in Ak

The development of spontaneous leukaemia was not retarded nor nns thn
me^ence lowered in tolerant Ak mice given repeated injectioroJIwI oj
spleen cells from lon^-leukaemic C3H mice. A retarding effect of CSH marrow
on tlie development of spontaneous lymphomas in (AIIR x C3H)F, hvbrids lias
been reported b 3 ^ Lorenz, Law and Congdon, (1954). It is possible that this effect
IS not demonstrable in the pure strain.

The occurrence of salivary and other tumours in tolerant C3H mice and of
mammary tumours in tolerant Ak mice was unexpected. Only Ak females tliat
had received C3H and not C3Hf ceils developed breast tumours, whicli siwgesf.s
transfer of the Bittner agent at birth via the cells. The C3Hf mice that dei'elopcd
salivary and other tumours all came from two litters injected on the same dav
with the Same preparation of norma] Ak cells ; all the surviving members of t!ic
two litters were affected. None received leukaemogenic filtrate. This particular
distribution, the variety of the tumours, and the fact that the animals had all
been injected on the first day of life with the same preparation of cells strongly
suggests that the cells were obtained from Ak mice carrying the polyoma virus
kno^vn to be present in various Ak stocks (Rowe e/ ah, 1959). Neither tissue
culture (Stewart et al., 1957) nor lugh speed centrifugation (Buffett el ah, 195S)
is thus necessary^ to disclose the multipotentiality of this agent.

SUaEMAEV

1. Acquired tolerance of C3H and Ak thjmuis homografts has been achieved
in Ak and CSH mice, respectively, by the intravenous injection at birth of C3H
and Ak thjnnus or spleen cells.

2. Lymphocytic neoplasms developed in Ale thy^muses grafted to thymectomized
tolerant C3H mice. None were, however, seen in CSH thymuses grafted to thy-
mectomized tolerant Ak mice.

3. The IjTtnphocytic neoplasms arising in tolerant C3H mice were in some

cases transplantable only to Ak mice, in others to botli Ak and non-toleraiif
adult CSH, and in others only to non-tolerant CSH. r ti.

4 Three out of seven Ijunphoid tumours developing at the site of the .Ak
thymus graft in tolerant C3H mice completely regressed after treatment of tlm
host with C3H lymphoid cells from mice previously sensitized against noriiuii

other tumours occurred in these experiments. Tolemnt Ak femsic mice
developed manimar,. carcinomas. A group of toler.rat CSH "f

birth with the same preparation of normal Ale cells dm eloped nuiltij . < ■ .

gland tumours and other tumours.

T ndsh to thank Professor P. B. Medawar, F.R.S. for reading the .script and for
hislaluabJe advice and criticism, and Dr. L. Brent

niques of intravenous injection of new-born mice and of ^1'"’ -• ;

thanks are due to Dr. N. F. C. Govdng of the

Royml Marsden Hospitai, for Ins help m diagnosin . ^

Sutton of the Department of 3Iorbrd f f lam

Medical School, for his description of the j! f. Kollcr for t’.eir

indebted to Professor A. Haddow, F.R-S. and to i roiCrim

THYMUS HOMOGRAFTS IK l^tMUKOLOGICALLY TOLERANT MICE

interest throughout this work and to the Gaggin Research Fellowsliijr of tlio
University of Queensland, Brisbane, Australia for its invaluable suirjiort,. This
investigation has been supported bj' grants to the Chester Beatty Research
Institute (Institute of Cancer Research ; Royal Cancer Hosjhtal) from the itledical
Research Council, the British Empire Cancer Cam])aign, the Jane Collin Cliilds
Memorial Fund for Medical Research, the Anna Fuller Fund and the Nnt.ionnl
Cancer Institute of the National Institutes of Health, U.S. Public Health Service.

REFERENCES

BiLmoHAM, R. E. Aim Brent, L.— (19.56) Proc. Pmi. Soc. B, 146, 78.— (1959) Phil.
Trans. B. 242, 439.

Mm, Brent, L. and Medavvar, P. B,— (1953) Nrilurc, 172, 603.— (1954) Proc. Rnu.

Soc. B, 143, 58.— (1956) Phil. Trnns., B, 239, 357.

Mm AND Medawar, P. B.— (1951) J. exp. Biol., 28, 385.

Brent, L.--(1959) ‘ Tools of Biological Research ’, p. .57, H. J. B. Atkins, cd. O.xford
(Blackwell).

G.-(1952) Acta Un. int. Cancr., 7, 761.
w, R. F. Cojemerford, S. L., Forth, J. and Hunter, M. J,— (]95S) Proc.
i>oc.exp.B 2 oL,iy.y., 99 , 401 .

N.— (1944) Cancer Res.. 4. 1.

Cl Proc. Soc. exp. Biol., N.Y., 100, 325.

^(‘(■Cancer Inst., n, S3.

CoS H ®--(1956) Cancer Res., 16, 434.

12 789.-(1957) Ann. N.Y. Acad. Sci., 68, 616.
andR^S H- 253.— (lOoOt) Ibid., 11, 425.

iWlO.^’’ J.— (1959) Proc. Soc. exp. Biol, N.Y.

,, ' ISbG.Tw Wo1Sen?nr°°''i C C.-(1954) Ciba Symp., Leukaemia Research,
M-mnowELL E f t S ’ ^'l- Lo^^don (Churchill).

N \1anL^T^™''’w’ ^'^''arnick, M., Taylor,

,, „ 37 47 ■’ aud Winterstein, M. P._(1938) Yearb. Ca

‘^k'ExDY D p '-r’'

fc-tiSH, d, Smra J M 'i'vtf? f Cancer Jfes.. 4, 377.

»• Ku’se“” ?°r' T'‘- =«•

•ueler, j. P — (19o8) Immunology, 1, 1.

lS09.-,.360„)

“’^'■Vl’S-TAvxiR A? f"'^''‘\171. 267.-(1954) Proc. Roy. Soc., B 142 72
Ho've, W ? H ' E. C.-(1938) Proc. Soc. exp. Bid., N. Y.,

R. J.-(1959) J. exp. iled.,

E- Blackva-ell, R. H.-(1959) ./. nat.

Mnn, ®oi^gese, N. G. and Grubbs, G. E.—

I'''"' I- 15, 842.

N.r,., 7, 211.

Carneg.

•n07«4 n Y' ^ orn. .j . piast.

GJo8) QuaH. .J. exp. Physiol., 43, 91.

25C

JER K. MODY

From, the Department of Experimental Pathology and Cancer Besearch,

U mversiiy of Leedsf

Received for publication SInrch 12, 1960

The present experiments are concerned witli the state of tlie ovaries, incliitliiiv
the process of tumour formation, foUowing skin applications of four chemical
carcinogens to virgin imce of the IP strain. Howell, Merchant and Orr (1 9,^.4)
s lowed that skin applications of 9 : 10-dimethjd-l : 2-benzanthracene induced a
liigli incidence of ovarian tumours in inbred or li^'^drid virgin mice of this strain
as compared vdth three other inbred strains. Merchant (1957) also tested 20-
methylcholanthrene in a small number of mice of IF origin and obtained a few
microscopical tumours. In a previous paper (Mody, 1960) the normal virgin IF
ovary at various ages was described and it was shown that frequent spontaneous
pseudopregnancy occurs in grouped virgins.

JUTERIAL AND METHODS

IF virgin females, approximately sixteen weeks old, were subjected to four
fortnightly skin applications of an 0'5 per cent solution of a cliemical carcinogen
(obtained from L. Light & Go. Ltd., Colnbrook) in arachis oil. At each painting
1 ml. of the solution was applied as 8 drops to the dorsal and S drops to the ventral
side of the entire trunk surface. The animals in each group were Idlled in batches
at 0-3, 4-7, 8-11, and 12-15 weeks from the date of commencement of painting
and from then onwards at S-weeklj’’ intervals until 70 weeks. The organs were
examined and the tissues fixed, cut and stained as described Mody (1000).
Between 8 and 12 weeks after the start of treatment a batch of 4 mice from each
group was used for a dail5'' tliree-week study of the vaginal smear.

The groups comprised ;

(i) Sixty females treated vdth 9 : 10-dimethyl- 1 : 2-bonzanthraccne
(DMB).

(ii) Fifty-three females treated with 3 : 4-benzpjTene (BP).

(iii) Thirtj^-seven females treated with 20-meth3'lcho!anthrenc (M(-).

(iv) Sixty females treated with 1:2:5: G-dibenzantiiraccne (DB).

RESULTS

Incidence and Age of Appearance of Ovarian Tumours

Table I shows the number of tumours obtained with four
the age being counted from the date of the first paintmg. The f umoun, rau,_(

* Part of a thesis presented for the degree of P5.D. of ' j n

f Permanent address : Indian Cancer Research Centre. P . < . -<

IKDUCED OVAKIAX TUMOUUS IX IE IMICE

from microscopical size to 1 cm. or more in dinmctcr. I'welve l.iunouns were
obtained in DMB-treated mice, 4 in BP-treated and a donbtfid early tnmoiir in
MC-treated, while none occurred in the ])B-gronp. In addition, early tvimonis
which could not be identified with certainty occurred in 4 DMB- and 4 BP-treated
mice. All the tumours occurred prior to 52 weeks from the coiumencemcnt of
treatment.

Post-mortem Ajipcaramc of the Ovaries
At autopsy the treated ovaries were either normal, enlarged or reduced in
size, the ovaries on the two sides being often of different sizes. On the whole t he
ovaries appeared normal to the naked eye until tV.l weeks from the start of tTcat^-
ment and from then on showed varying degrees of enlargement culminating in
haemorrhagic or non-haemorrhagic cysts or tumours : at later age.s many ovaries
were small and shrunken. In the DB-treated mice, however, the ovaries showed
no effect of the paintings but underwent a rednetion in size with advancing ago
comparable to that described for untreated mice (Mody, 19(50).

Cysts and tumours were always unilateral and the o]r]iositc ovaries were
reduced or normal in size. Tumorous ovaries were observed with the naked eye
only in DMB- and BP-treated mice. Seven DitIB-treated mice had grossly
detectable tumours between 16 and 51 weeks, 6 being in the right ovaiy. The
tumours ranged from 7 mm. to 2 cm. in diameter and were fleshy ]iink. grey or
jellowish-white in colour with darker areas of haemorrhage. I’lie surface was
smooth and slightly lobulated, the consistency soft and there were some
lomorrhagic cysts. The larger tumours had loose fibrous adhesions with the
pen oneal wall. In BP-treated mice two tumours were observed with the naked
wiL 'W’eeks was l-o cm. in diameter, yellowish-white in colour,

surface and free from adhesions, while the other occurring at 31
■SA\as only just recognisable and was greynsh and slightly raised.

Histology of the Tumours

classified as shown in Table I ; granulosa cell tjqoe (12),
gfanulosa granulosa cell type (2) and mixed thecal, luteinised and

addition one luteoma was induced by BP. The
'‘'Hitiou to the naked eye were all of granulosa cell type. In

’'’iehthavou ^ tumours, there were very early lesions in 9 ovaries which
^ been tumorous but were nob certainly so.

f a compact pseudofollicular pattern. The pseudo-
resembled small anovular follicles. The central lumen
ain some eosinophilic material, the latter type resembl-
. Some of the granulosa cells composing the tumours
shape and showed mitosis, but most of the cells were
(^^g- t)- Some tumours contained
he cysts were either large blood loculi, empty spaces
fluid. All the tumours were free from lipochrome
e were small amounts of iron pigment in haemorrhagic
umours were usually undifferentiated.

follio

in .'?, 01

«reas.

258

JER K. JIODY

C5
+
CjkCI
+ +

INDUCED OVAKIAN TUMOURS IX IE MICE

250

Mixed tumonrs „

Tliese were microscopical in size. In addition to granulosa cells, they con-
tained luteinised cells (Fig. 2) and even thecal cells, the latter two coll types
shomng no mitoses.

Lukomas

The one tumour of this typo was microscopical in size and contained a central
organising haemorrhage. The component cells rcscinhlcd normal lutein colls,
but were more variable in size and had irregular nuclei, rarely in mitosis (Fig, A).

One-generation transplantation of a qranulosn cell tumour

The single large granulosa cell tumour induced hy BP was trans))lantcd std)-
cutaneously into 6 mature IF male mice, in all of which jmljmblc t.nmonr.s about
2 cm. in diameter developed by lG-10 weeks. Histologically the grafted tumouns
resembled the original in structure. The mammary glands of the tumour-hearing
males showed lobular development while there was atrojrhy of the seminiferous
tubules and lack of secretion in the seminal vosiclc.s. These changes were
regarded as due to hormone secretion by the transplant.

A. Processes Leading to Tumour Formation (DMB, BP, or MO)

These may be described under the following headings (Fig. 4) :

Stage 1. Total loss of follicles.

Stage 2. Luteinisation.

Stage 3. Prominence of the germinal epitlielium.

Stage 4. Occurrence of nodules.

Stage 5. Occurrence of tumours.

exiIf**Vw* follicles . — This is brought about by degeneration of all the

''g ollicles and failure of further formation of follicles. The earliest degencra-

Tre^doyarj- (DMB, BP or MC)

Degeneration of follicles

Fig. 4._

Jlerging of corpora luten

Breaking awa3’ of tho thecae
^Proliferation and luteinisation of the thecae

(Thecal luteinisation or luteomatous change)

i'.

Occurrence of small nodules in thecal luteinisation

. 1 '

Bigger diffuse nodules
(Early foci of granulosa cell tumours)

1 ^

Granulosa cell tumours

Sequence of changes leading to granulosa cell

tumours.

262

JER K. MODY

fjfoVigeOl '‘” 7 ^ Sfceadilj^ replace tlie cells responsible for luteinisskii

(iiodj , 1960). In the degenerated Intern cells lipochrome pigment is seen wliici)

7 T pliagocjdes. Cystic ovaries may he found occasionnllvf

In all the 12 mice bearing unilateral DMB-induced tumours of the »raniilo<n
cell senes the contra ateral ovarj^ was atrophic. Unilateral or bilateral atronl.'ir '
changes Avere studied in 25 DMB non -tumour-bearing nuce, starting as carlv ,•
10 ireeks after the beginning of treatment. In BB-treated mice all 4 contralatenh
ovaries of the tumour bearing mice ivere atrophic. In 21 of the non-tummir>
bearing mice treated with BP for over 16 iveeks there v'ere atrophic chnnge.s.'

EXPLANATION OF PLATES

Fig. 1. — Part of a large granulosa cell tumour of compact pseudofolJiciiJnr pattern. A large
oj’stic space containing fluid is seen towards the periphery. Tiiirtv-five weeks after start of
DJIB treatment. x90.

Fig. 2. — Part of an early mL\'ed tumour composed of luteinised and granulosa cells. The
cjdoplasm in the luteinised cells is more abundant and pale staining. Fourteen weeks after
the start of D5IB treatment, x 60.

Fig. 3. — Part of a luteoma shorving cell cords similar to tiie lutein cells of the corpus lutenm
but with a greater degree of variation in size and shape of the nuclei. Thirty weeks after
start of BP treatment, x 375.

Fig. 5 . — Stage of follicle degeneration. The compact dark outermost granulosa cell layer is
particularly distinct in the large follicle (centre right) which is undergoing degeneration.
No degenerating cells are present in the surrounding thecae. Atretic remnants and engorget!
capillaries are seen. Towards bottom left are corpora lutea. Three weeks after start of
D5IB treatment, x 90.

Fig. 6. — Earliest degenerative changes seen in the ovum. Disintegration of the nucleus and fat
droplets in the cytopiasm. Three weeks after start of DMB treatment. x340.

Fig. 7. — ^An aggregate of large pale theea-tj'pe ceils in subgerminui position and clo.se to an
atretic follicular remnant. Some of the cells are in mitosis. Three a-eeks after start of D.MH

trea tment. x 285.

Fig. S. — Total loss of follicles. Atretic remnants (AB) and diffuse iuteinifjntion. due to pre-
mature merging of coipora lutea, can be seen. A tiny nodule ) towards toj) centre in an area
of iuteinisation. Eleven weeks after start of DMB treatment. x70.

Fig. 9. — Two mitotic figures among luteinised cells, probably an area of early thecal hiteiiiHa-
tion. Three weeks after start of DMB treatment. x5Co. . . , ii i

Fig. 10.— A well-marked nodule towards top right in an area of Iuteinisation. A capiflory ana
a I^unphatic vessel are associated with it. AVidespread Iuteinisation and seatterer irupt
spaces. Elet'en weeks after start of DMB treatment, x /5. i ;

Fig. II.— a nodule surrounded bv engorged capillaries in an area of Iuteinisation. Die niiri
of the cells within the nodule are closely packed and variable in .s^e, shape imd slainine.
A prominent nucleolus is often present. Eleven -weeks after start of mon . . *

Fig. I2---IVO ill-deimed nodules within (in area of lutemjsation. *

similar to those of ^anulosa cell tumours. Thirty-five weeks after s art o

Fig. 14. — Non-tumorous atrophy, x 4«v ^

lium, especially towards extreme right and “I"”*?® ""“''V "'^nemrnd Intern cell towariN

epithelial cells towards the peripliery. Pale pigment -laden degenerated lutein

centre. Fifty weeks after start of DMB treatment, x 8a. undergoing ntr.-.i...

Fjo. 15.— a large number of intact coqjora lutea. , 'foB (.entmant. (Compw

No eifect of the treatment visible. Fourteen weeks after start o ^
with Fig. 7 and 9 from DMB treated mice m the same age group.) / 3 ■

British JorRXAi. or Cantiiii.

Vnl. NIV. No

^locly.

BnmsH or Cakcek.

V»,V MV, N" ‘

Mody.

kduced ovabiak tumours in ir mice

263

including 5 cystic right ovane^ fibrous scars together

in these JlC-treated ovaries.

C. Processes Leading to Senile Atrophy

The ovaries ^

™riy ^“rSKncStoik liaoi’cTable m). GtaaBan fomdea we somewhat

Table III.— ilficroscoiu'cat Appearances of DB-treated Ovanes

Germinal epithelium
Follicles

Corporal lutea

Survival following start
of treatment*

Xumber of mice
Anovular buds
Dark staining cells
Primordial
Graafian
Atretic follicles
Atretic remnants
New
Old

Degenerating (early)
Degenerated
Pigment
Fibrous scars

O-lo

16-33

. 34-51 .

-r

-4-

. -r-i- •

4-4-+ .

-r

. +4- •

—

. 4-4-4- *

—

4--r-!-

4- 4“ 4“

. —

4" 4" 4-

-1-

• -1- -rp- + •

-b+'+-f •

—

(Compare with Table I, Mody, 1960.)

>52

-r +

4 -+

+ + +
+ + +
+ + + +
+

more frequent at older ages than in normal ovaries of the same ages. Cystic
follicles without haemorrhage, anovular follicles and corpora lutea atretica, i.e.
atretic follicles containing lipoids (Fekete, 1946), were occasional!}’’ observed while
they were rarely observed in normal ovaries. Intact young and old corpora
lutea persisted until about 30 weeks after treatment i.e. until 46 weeks of age.
Involution commenced within 4 weeks follotving the beginning of treatment and
the content of degenerating (those undergoing early degeneration) and completely
degenerated corpora lutea was greater than in normal ovaries, where more lipo-
chromc pigment was present. The old DB-treated ovaries showed fibrous Scarring
and hyaline degeneration in the avails of small arterioles, these changes being
\nicnmmon in normal ovaries. Thus after DB treatment, total loss of follicles
and difTu.«c and thecal luteinisation were absent (Fig. 4). Prominence of the
germinal cjiithelium was noted with age but proliferation and invaginations were
Css <‘'’><hnt than m normal ovaries. Senile atrophy was seen in 20 of the mice

.Ct “"e '“ger

Uterus

tin microscopical cpmination, the state of the uterus was classed as atror^hc
norin.il or liaving cystic hvnemlasia Tn fin Tiam + x i . trophic,

ven^ pn^ent in and cS ®^IB-treated mice, ovarian tumours

eontrLi L, n; “ h}'perplasia occurred m 5 of these (Table Tt^r

b. "hr

264

JER K. MODY

c 5 '’Stic lijTierplasia. Wien the size of the ovarian tumour ivas considered, it was
found that the largest were often not accompanied bj’^ cystic Inperplnsia. Bali

Table IV. — State of the Uterus and Distribution of Breast Tumours i
Ovarian Tumour-hearing and Non-tumour-bearing Mice

Ovarian tumour present

!?!

Ovarian tumour absent

I. DMB

II. BP

in. MC :

. 1

. 5

. -

. 3

. -

. 3

. 5

. 3

IV. on

Cystic hyperplasia
Cystic hj-perplasia
+ breast tumour
Iformal uterus
+ breast tumour
Normal uterus

without breast tiunour

Cystic hj'perplasia
Cystic h3'perplasia
' + breast tumour
Normal uterus

+ breast tumour r 3 .1

Atrophy of uterus • “ ■ *’ • j, '

Atrophy of uterus . - ■ ~ •

-f- breast tumour

Vaginal Smears

In vie. of the o-urrence °f

virgins kept in fours, a 3-week study of the ^ ^ Great individual

groups of treated mice 2 among DMB--

Liation and irregularity m the length of Jhe C3 ^ By contra..!.

MC- and DB-treated mice ^lo-trus i^as ^ , ,„y,

in BP-treated mice a short at dioestrus. The cycle ni Bl -

and there was no ^ that of normal virgins and similar

treated mice is thus * Further evidence is nccessaiy to sub-

to that of anosmic mice (Mod}, luouj.

stantiate this.

Mammarij Ttmours in !'

Of 60 DMB-treated mice, ^^tlS aSiw of Ovarian tumoims in

that DMB-induced ovarian and breast tun
being random.

discussion

Incidence of ovarian tumours , 5 ug trcatmmit wh j

induced OVAEIAK TIBIOUBS IK IE MICE

265

Sequence of ovarian changes , i • •

The first ovarian effect of DJIB, BP or MC is damage to the ovum and is
followed by degeneration of all the existing follicles and fedure of new follicles
to develop. These effects are rather more rapid mth DlIB than vnth the other
two chemicals. Abnormalities of luteinisation are followed by the appearance of
nodules (Fig. 9 and 10). These are derived from theca-lutein cells and are thought
to be the starting point of the tumours of the granulosa cell series. These nodules
may be bilateral, although the tumours are always unilateral, and it is therefore
suggested that some regress. The final stage in the development of the nodules
into tumours was seen after DMB and BP treatment but was not reached after
JIG treatment in these experiments. From histological examination it is not
possible to be certain when the growth of the nodules becomes autonomous. If
nodules fail to develop, or regress, the ovaries undergo regressive changes charac-
terised chiefly by proliferation of the cells of the germinal epithelium, which stream
inwards to replace the lutein tissue.

By contrast, the changes observed after DB treatment resemble those seen in
ageing normal virgins. Total loss of follicles did not occur, abnormal types of
luteinisation and nodules were absent and there were no tumours. The specificity
of action on the ovary of these chemicals, all of which are carcinogenic to other
organs (c.g. the sldn), is thus apparent.

Tlie histogenesis of the tumours induced by DjMB and BP is similar to that
seen in irradiated ovaries (Brambell and Parkes, 1927 ; Giest, Gaines and
Pollack, 1939) and in intrasplenic ovarian grafts (Biskind and Biskind, 1949).
1 he sequence of events after chemicals may include a transitional luteomatous
stage but this phase seems to be less persistent than that observed by Lipschutz
(19(10) and Lipschutz, Rojas, Cerisola and Iglesias (1960) in intrasplenic or
frapncntcd ovaries. From the present experiments it appears that granulosa
cell tumours can arise from areas of abnormal luteinisation, mthout an inter-
vening luteomatous phase, but this is not certain.

Jtehlion between the occurrence of ovarian and breast tumours

1 DMB, ovarian and breast tumours frequently occurred in the same

uouse m lore was no such association when the carcinogen was BP MG or

( uin(.iir.s when grafted into normal mice. develop ovanan

Ihnn-nml effect, of the ovarian tumours

ir'irutrsVw^^^^^^^^^ ^--re 5 in which cystic

ioraitx:™:

266

JER K. MODY

seen in iobular dTvX^Vtlntirbt^ “SSe.S!

and lack of secretion in the seminal vesicles. ^

SUMMARY

Limited skin applications of four carcinogens (DJIB, BP, MC and DB) were
made to inbred virgin IF mice, which were subsequentlj’- killed at a^cs raiwiiw
from 16 to 70 weeks in order that the sequence of ovarian changes micrlif bo
studied. °

Ovarian tumours of the granulosa-cell series ivere induced by means of BMB
and BP, but not vdth MC or DE. The induction period was shorter with DMB.
Pre-tumorous changes were induced udth MC, but DB exerted no effect upon the
ovary.

The tumours were unilateral and of the granulosa cell series, the granuIo.s.i
cell type being predominant. They resembled those occurring spontancoiisly
in some strains of mice, those induced b}'- X-irradiation or in intrasplenic ovarin'r
grafts in castrates and the granulosa cell tumours of the human ovar}'.

The sequence of histological changes in the ovary after treatment with D.MB,
BP or MC is death of the ova and degeneration of all the follicles, failure of new
follicles to develop, merging of the corpora lutea, proliferation and lutcini.sation
of theca cells and formation of multifocal nodules from these luteinised theca
cells in one or both ovaries. The tumours arise unilaterally in one or more
nodules, the remainder of which undergo regression. The secondarj' proliferation
and luteinisation of the theca cells following merging of the corpora lutea, with
subsequent nodule formation, is regarded as the essential precursor of tumour
formation. The ovary contralateral to the tumour-bearing ovap’-, or both ovario.-(
where no tumour is present, undergoes reduction in size. This is characteri.scd by
a streaming into the substance of the ovary of dark staining cells derived from the
germinal epithelium and the accumulation of lipochrome pigment in phagocytes.

Following treatment with DB the normal age changes which take place m
\Trgins occur (Mody, 1960). Large cystic follicles, persistent corpora hitca,
fibrous scars and hyaline degeneration of blood vessel walls are more frequent
than in the normal and some ovaries become greatly reduced in size.

REFERENCES

Bau, T. ard Furth, J.— (1949) Cancer Bes., 9, 449

Howuk, J. S., Makchant, J. and Orb, J. W--(19o4) Br,t. J. Cancer. 8, Ma.
Lipschutz, A.-(1960) Ada.Un. ini Cancr., 16, 149-
Idem, Rojas, G.. Cerisola, H. and IgRESias,

SIarchant, j. — (1957) Brit. J. Cancer, 11, ( > »■ ) - jy^cds University.

Mody, j.— (1960) Thesis presented for the degree of Ph.D. ot Leeds on j

“SSTlS” c!i

B. D. PULLINGER and S. IVERSEN

ft. C.«.r S^mrck »>»»' B."'*”"

Glasgota, C.3.

Received for publication February' 1C, lOGO

Befekekce data in respect of age, number of litters and mammary carcinoma
• ■ 1 HOP in r TT /He and RIII//Pu mice have been recorded and analysed quanti-

ofXtual con.pae.on noth .esnlt, from
ovarian hormones. The incidence of tumours of other sites is included.

5IATEBIALS AND JEETHODS

Origin of mouse strains

Some particulars of the first 34 generations after cross-suckling 2 females and
1 male of an RIII litter comprising 482 former breeding females have previously
been reported (Pullinger, 1952a, 1955). Absence of evidence of mammary
tumour agent from extracts of 2 tumours derived from the cross-suckied strain
and tested in susceptible agent-free P.l hybrids of C57 mothers and Rlllb fathers,
together with an overall reduction in mammary carcinoma from 80 to less than
3 per cent in breeders and from 69 per cent to nil in virgin females through 34
generations allowed the presumption that the agent had been excluded from all
sublines. The present report concerns generations 35 to 52 since cross-suckling.
The number of litters a female was allowed to bear was deliberately limited to
3 in the majority of breeders in F.40 and to 6 in F.41 to F.44 but in all others
breeding was unrestricted (Table I) and was interrupted only for the purposes of
securing the next generation or sufficient animals for experiment. Twenty-four
breeders only were self-limited to one or two litters.

Progeny of C3H, mice were derived from a litter in the F.23 generation which
was given to this hospital in 1954 by Dr. W. E. Heston. This substrain was
derived by Caesarian section and cross-suckling from Andervont’s C3H line
(Anderyont and McEleney, 1941) in which mammary tumour incidence was higher
in virgin females than in breeders. Progeny of the cross-suckled C,H,/He sub-
strams were exhaustively tested for erddence of agent and none was found bv
Heston and Ins colleagues (Heston el al., 1950 ; Heston and Deringer 1952 •

these laboratories has been carried out by brother and sister matings supervfsed

UrPP r one of the authors. With the exception of 17 out of 108 females

breeding was unlimited and was interrunted bv removal nf + r i

Sur^L7tTt£ teXg^ox^^^^^ ''

ceased breedlng™t7lmilVfOTl^^^^^^^ had

268

B. D. PULLINGEE AND g, D'ERSEN

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TUMOUK IKCIBENCE IN MICE

I’m)

Both ottataB we housed in the same room in 8“''';';™^,;™ 'X’

^vith mesh lids and sawdust and wood shavings. 1' ood in the lonn ol (,n m
of comnosition 41 (of the Medical Research Council’s Laboratory Aniinals Centre
anrShigVati were supplied ad libiUm. Every three months ior a pono

o?3 weeks streptomycin, 0-025 per cent, was added to j’^'c H 1110" h
epidemics of Tyzzer’s disease. Six months after arrival of tlic C3H/ litter the
rlom was air-conditioned udth an electrostatic precipitator to reduce atmospheric
pollution for other purposes and was kept at 78-80“ F All animals were cxaininecl
weekly for tumours or other disease. They were allowed to live out. tlicir In c.s
and were killed only when moribund or unable to feed or drink or when a tninonr
had developed. Tissues for microscopic examination and for bulk-staining were
fixed as a routine in Bonin’s fluid or in other fixatives as stated. A feu of t he
more dense mammary adenomas (hyperplastic nodules) were examined micro-
scopically. In this way some presumed early carcinomas were detected but
because all such nodules were not examined, none has been included among the
gross, palpable tumours upon which incidence is customarily based. Grafts of
some tumours were made in males and females of their respective strains or in
F.l hybrids. Biopsies were done on a sample of tumour bearers. The incidence
of other tumours is recorded with the exception of the lymphoblastoma group in
C3H/.

RESULTS

Rill,

The overall incidence amounted to 14 mammary carcinomas in 544 breedens
in 18 generations (Table I). The 14 RIII/ mammary tumours were less readily
typed according to the description by Dunn (1959) than were those in C3H, mice.
Type A, of uniform fine acinar structure, and Type B, a group of diverse acinar,
cystic and papillary formations, merged more often. As observed by Foulds
(1956) compound organoid carcinoma was relatively common. With these
reservations there were 5 malignant adenoacanthoraas (1 organoid), 2 anaplastic
carcinomas, 4 type B tumours and 3 compound organoid carcinomas without
squamous change. Of the total RIII; mammary tumours seen since cross-
suckling, amongst 1026 breeders 21 were in anterior and 5 in posterior nipple
areas, a distribution consistent with that of RIII^ adenomas (Pullinger lOsHi)
caremoma in C,H/ females (Prehn, Main aU SchLiderman
1954 . The latter authors found also that the degree of unevenness of dktSfnn
was largely a function of tumour age The greater the dfribution

rela^nsli^^d^Lta^pS^^^^^^^^^^ ^3’ the present authors this

celled ;ro^s?a? at'tukSf '"3“ Whoblastoma, reticulum-

98 per eenl whereL in p fto 14 them I’.44 it was

change in anatomical distribution was as str^ra'^^tf “ ^"®^ders. The

In early generations regional and abdominal IW, t “ incidence,

only -6 per cent of lesions occurred in liver or sS^ were mainly aifected ;
88 per cent of all these tumours affected Se laUe™

^ >nain tj-pes of these liver lesions were seen

seen, a neoplastic extramedullary

270

s. D. PULLINGER and s.

lYERSEK

Tabm ll-miMon of SpcMa,>eom Corchco Jwi,,

10 Age, Strain and Site

Age in months

Strain Nipple areas

Kill/ . Anterior .2024Iinnnooon Totnh

Posterior ■ 0 0 0 0 0 f ^ f f ^ U f ^ ^ ^ ^ ^ S H

C,H

'3*1/ . Anterior

Posterior

”^ 00101301200321221 “' “>■'

00000000000220000020 1

erjijlrropoesis and po]ymorpIiic reticulum-ceUed groiidhs. TJiis large increase in
a more lethal type of tumour than are ] 3 unph node lesions might liave reduced
the average survival age to below that at rvhich manimarj^ carcinoma woidd arise
but this was not so. The average age at death of the first 4S2 breeders in F.1-31
was 20 months and the average tumour age was 1 9 montlis. In F.35 to 52 genera-
tions comprising 544 breeders the average tumour age was 19-G months, and
sundval age 19-4 months.

Fewer tumours of other sites were observed and usuall}' at a later age with tlie
exception of some sarcomas (Table I). The earliest of these, two osteogenic
sarcomas of bone, were found in two 7 month old mice. Bone tumours were found
slightly less often than mammary carcinoma and occurred at random, only
occasionally shoving familial relationships. Three which arose in F.Gl were nil
descended from a common grandmother in F.49. This female developed n
mandibular carcinoma at 27 months of age, containing hair shafts similar to
tumours described by van Rijssel and Miihlbock (1955). In F,39 a brother and
sister had osteogenic sarcomas, the female in the right femur, the male in the right
foreleg above the paw. The common ancestor, without this tumour, of all in
Table I with osteogenic sarcoma, belonged to P.32. No other near relationsiiifts
were seen. Though relatively few males were kept to old age the predominance
of bone tumours in females noted in Simpson mice by Py'bus and Miller (1940)
was less striking in the RIII^ strain. One osteogenic sarcoma in 100 Hill/
male breeders of the same generations was observed but others were found in
males set aside for experiments unconnected wdth induction of tumours.

Hepatoma was uncommon and none was seen before F.25 although lhc.se
growths had been sought. The usual preponderance in males over breeding
females was found but not over virgin females wluch had a similar incidence
(Table III). These results are referred to again with findings in the strain,
Intracytoplasmic inclusions which have been described by Head and Laird (lO.IO)
were found in all except one RIH/ hepatoma. An unusual group of growtln
occurred in the rectum in some breeders and virgin females. These were eitficr
carcinomas of rectal mucosa, sarcomas or rai.ved tumours invading the mu.sciila-
ture. Some were associated vdth cy'stic epithelium lyung between longitudirM
and circular muscle fibres. The same relationships may have been pre.^ent
others but serial sections were not made. One parotid tumour m a .

F.39, 1 among the progeny of females mated at random for prof^^ction o •.
mental animals, and 1 in an ovariectomised breeder mre lecn s | j |^.

before that generation. An epithelial tumour of subcutancou.s tis.Mic j.robal.ly

derived from epidermis was not classified.

MAMMAKY tumour INCIBENCR IN MICU

271

One hundred end eight feuralcs rvere fof

17 out of 108 u-os limited to 10 to U "'“■>«« “ “f 17 dovolo,,ed

i„ 0 reference group to be leeoi Wet MO to U,o prceeut .mulyrie nil

hi"”h«n “Steed together ns one group of uSSImI-' of

popnlntion at rish,_^-e.dyteo

t risk. iwenx.y-T;wu

Clippie areas and 6 in the 2 posterior pairs, a proportion l”6>'cr h'v... ' 7.

ponding with that found by Prehn, Mam and Schnc.dcr.nan (1!)..4 ■ 11 •

Of Type A (uniformly acinar) there were 11 examples, of Type B ( nultifon
acinar!^ cystic and papillary) there were 12, and of Type C, composed of Mim

acinar, cystic and papillary) there were ana oi rypo e n.,. .... ... ........

uniform epithelial-lined cysts enclosed in layers of spindle cells, flierc uere
One malignant adenoacanthoma and 2 carcinosarcomas were diagnosed .

None of the 28 mammary tumours was associated with pituitary enlargement
or adrenal cortical carcinoma. Two were associated noth small ovarian grnnulo.sa-
celled tumours. Forty-six pairs of adrenal glands were examined micr()sco[)ienlly.
Proliferation of subcapsular A cells, usually fusiform with deejily st.ained nuclei
and scanty cytoplasm, and the change from lipoid to compact fasciculata cells
had occurred in all. Large rounded or polygonal vacuolated pale staining B
cells were found in clusters in the cortex of one or both of 18 pairs of adrenals and
ceroid (chromolipoid) in 11 pairs mainly in older animals. Cortical B cells were
found in 10 of 22 breeders rvith mammary carcinoma in 4 of which they were
hyperplastic ; they were found in 8 out of 24 witliout carcinoma and in 2 were
hyperplastic. Alphabetical tj^ping of abnormal adrenal cells is in accordance
wth the description of Woolley and Little (1945). The compact fasciculata cells
resembled those previously described in virgin GgH; females (Pullinger, I!):")!))
which ate found also in males. Three microscopic medullary adenomas, one
extracapsular adenoma of compact cells only and one of both A and compact cells
were found. These extracapsular nodules of compact cells can now he identified
as accessory adrenals which have undergone the same age changes as the adrenal
glands. Accessory mouse adrenals (described by Whitehead, 1932) have now
been found in C3H mice by Hummel (1958), Adrenal glands and nipple areas of
the same 18 females with, and of 16 without, mammarji^ carcinoma were examined
microscopically for correlations between the presence of B cortical cells and failure
of involution or of hyperplasia of mammary glands. No correlations were found.
Of / hepatomas 4 were associated with mammary carcinoma in breeding female.s.

poli^f of tumours were as follows : 5 ovarian granulosa-

celled or tubular adenomas often accompanied by cysts and 1 ovarian^ fibroma ■

1 St™* 1 • ‘ of a uterine Zn ’

™ found in ZtiiTreZllZIfZrt “f Z'
primary groirth elsewhere This animni v. i or other record of a

white nodules seen at necropsv in the InnL ^ mammary tumour and tlie
were bony structures. Mesenteric disease^nfT^^^ thought to be metastases but
strain (Simonds, 1925 ; Dunn, 1953) was comlm^ characteristic for the

Of »»W. Kill, and

(b.r,«) and added by AZraZZlZlS

er U90Z). ihe figures in Table III

272

B. D. PULLINGER AND S. IVERSEN

mcWenoe in vi^i„ fe„£ h nearerf thS in LS”™

Strain, sex
and parity
of mice

CjH/ Females :
Breeders
Virgins
Males ,

RIII/ Females :
Breeders
Virgins
Males .

Table III. — Incidence of Hepatoma

Mice alive

Number

at 1 6 months

with

and over

hepatoma

103

at 15 months

and over

419

Percentage

n-ith

hejintomn

G-S

24-5

29-8

0-93

9-4

S-S

No intracytoplasmic inclusions have been found in any of the C 3 E, iiepatomn?.
Hepatoma and hepatic reticulum-ceJled tumours were found together in 2 RIII,
animals. Without microscopic examination the liver-celled growth miglit have
been missed. The deeply groved channels in which their surface blood vessels
lie draw attention to the presence of hepatomas either alone or when combined
with l 3 Tnphoblastoma.

Second 'primary mammary carcinomas and grafts

The simultaneous appearance of more than one primar}' mammary tumour
when associated avith milk factor is common. Several were reported by Heston
et al. (1950) in females. None was seen among 28 tumour bearers here
recorded but in 4 out of 10 of the latter which lived the same length of time or
less than the remaining 6, a second primary' mammary' carcinoma was found at
49, 67, 74 and 79 days after excision of the original primary'. Tiie appearance or
non-appearance of a second tumour was unrelated to the number of hyperplastic
(adenomatous) nodules in mammary glands. No nodules were found in one
mouse with a second tumour and the average numbers in the 2 groups were
similar. Eecurrences of primary' grou'ths occurred in 8 out of 10, pulmonary
metastases in 2 of the 8.

First generation grafts of mammary carcinoma were made into or F.l
hybrid mice, 4 into males only, 13 into males and females and 1 into females only.
By chance the last was a C tumour, a type which rarely takes. Grafts wfiicli
reproduced the distinctive morphology' of the latter tumour grew and were pa p-
able in 3 months in all 4 tests females. Fifteen of the 17 grafts made in riialw
grew in all grafted animals. In one of the 17, one graft out of 3 had not grown m
2 months when the mice were Idlled. All of 3 grafts from another tumour ai c'
to grow in males in 7 months but the grafted sites were found at necropsy. Sec-
tions revealed apparently viable adenocarcinoma cells and tubu/c.s in dcii-e
collagen in all three. Of 14 tumours grafted into females, one failed to grow m
2 of 3 hosts in 2 months but apparently viable cells were found m section.'- oJ i
grafted site. The latent period between grafting and growth of hr.-_t gencraUoi
SspLts of different C,H, enremomes end of different fr.,gn,e„t- ei

(* W t\iniour wlncn fi''**

to grow iir every grafted 1 t » Ucpatomas ‘ ; i,i,t<.roin-

irsr "'"

Amlysii 0/ brtedng remds mi «n' l.k.Hnl ,.s»i«sl

In Pig 1 the probits of the incidence i i the iniinber of 1UTnonr>

the ^r^ber of Iters. The « and n -h f

amongst the difference ^Jt^enU N* the number of animals ivitli

or re... ..v., o ^ >

'“T»e W ,1«0) dat. ooo'y- ^MiVlsi'S

t»'-" ■'■> •"■" "

“T 11 tm Bill, aeeet-tree mice given m Table V.

Table TV.—CJi, Mice

Number Number

of o(

litters females

(= n) (=Nd)

1 . 9

2 . U

3 . n

4 . 19

5 . 20

6 . 10

7 . 6

8 . 9

9 . 0

10 . 5

11 . 2

12 . 3

Number

Incidence

Avernpe

\^ith

of tumours

tumours

/ 100N*\

nge of iVd

(= N*)

- A'a )

(days)

2.72

221

27-3

258

1.7-8

314

20-0

322

40-0

343

33-3

307

07-7

428

3G0

20-0

377

100-0

499

33-3

443

Time of lumotir
npiH'iirmiee

(days)

“84

907

010. 730. 8.78
780. 847. 003
090, 732, 7.70, 808
730, 088. 807, 842
,714, 730

372, 472. 733, 774.
803, 870

.72!)

018, 070
070

108 28

As can be seen from this figure all the data conform to a straight line course
which suggests, as pointed out by Shimkin (1945) that “ the effect of pregnancj’
upon mammary carcinogenesis (in strain A) is logarithmic in its accruance ”.
It is thus the number of pregnancies a mouse has had which is thought to increase
the tumour risk, a point of view which also has been expressed by Miihlbock ( 1 950)
and by Heston (1958). Hig. 1 shows undoubtedly that the probits of N*/N,,
increase with increasing litter number, but it is noteworthy that the straight
lines in B, C and D, where the lines marked a represent the regression lines for the
probit and those marked b the regression lines for the working probits have
practically identical slopes. This would indicate that the increase in tumour
risk was constant and independent of the strain of mice, which is contrary to

274

B. B. PULLINGER ANB S. Bv^ERSEE

observed findings. It seems therefore likely that the linear course obtained when
the data are calculated and plotted as in Fig. 1 gives a sort of relative measurement
and is only indicative of a possible identical mechanism of carcinogenesis uhich
is independent of the strain and of the presence of the Bittner agent. In Fig, 2
the probit of the incidence (N*/N^) is plotted against the average age of tlie

Fig. l.-Ordmnte: Probits of incidence.
A • Strain A (Jones, 1940).

B ■ CiHf (Heston, 1958).

C: c'Hf, Table IV.

D: Klllf, Table V.

Abscis-sa : Number of litters.

• = IVorking probits.

Q = Probits.

■Ntunbet

litters

(=n)

8
9

16
17

MAMMrVUY TUMOUR IKClOKNCR IN' MU'V
Table V . — liJUi

Xumber

Xumlior

with

females

tnmonrs

(= rVd)

(= iV)

104

! 1

544

Incidence
of timmiir
10(t.V\

.Average

np' of Sit

V " ~'sVJ

(dnVBl

nil

147

1 • no

1.76

— ~

‘2«6

236

2- 88

2.76

277

2-. 33

302

2-33

318

8-33

392

3-70

3S6

3-70

441

8-33

4.79

JC-64

463

460

—

491

—

.726

njij-orooi-''
(iln\ A

7ul

330. 401. inc.

327

4''.-,. 490
563
4 2. 7
497

417. 477. r.l**

mice and, as can be seen, the points arc distributed rniulomly nrotind strai^dit
lines, which now have significantly different slopes. It would ihercrorc b(' even
more justifiable to suggest that the effect of age upon nminnmry carcinogcncsiH

100 200 300 400 500

average age EDAYSJ

Fig. 2.— Ordinate : Probit of incidence.
.^scisM : Average age in days.

• — Working probits.

O = Probits.

27G

B. D. PULBINGER AND S. H^ERSEN’

§100

p: 60

id
o 60

S 40

k
o

5 201

Rmf

O \ ^ .0

O •

o = BREEDERS
• = VIRGINS

C3Hf

O •

O ^9

j °l o j P . ! ■- * -

200 300 400 500 600 700

800 900 WOO

AGE IN DAYS

Fig. 3. — Ordinate: Percentage of survivors.
Abscissa : Age in days.

MAMMARY TUMOUR INCmEXCK IX MICE

the litter number for the C3H/ and RIII; strains, respectively, it can he .seen lliat.
the points in the two figures are distributed in diametrically opposite direetions.
In Fig. 4 they follow a domiward trend indicating a decrease in dealli age witli
increasing litter number, while in Fig. 5 the death age if anything increases with
increasing litter number. As the probits of the incidence give a straiglit line
course when plotted against the number of litters and when ])Iotted against age
the inference that can be drawn is that the probit jirocednrc indicates the existence
of an identical basic mechanism, but docs not permit conclusions ns to tlie efic'ct

6 18 63 45 55 104 47 43 43 24 27 27 12 17 8

of litter number
tumours.

and/or to the effect of age upon

the incidence

of mammary

SiranVLABY

of littem^achV^"^ breeding females have been observed Tl 1

mamman- ea ® o.nd her survival

mammarv f^ave been recorded. There were 2S ‘

n ammary carcinomas.

"icluded. 01 otner sites have beei

278

B. D. PULLINGEK AND S. IVEBSEN

REFERENCES

Agnbw, L. R. C. and Gaednee, W. U.— (1952) Cancer Bes. 12 757
Andebvont, H. B.~(19d0) J. mt. Cancer Insi., 11 581 ’ '

Idem AND Dunn, T. B.~(1952) Ibid., 13, 455.

Idem- AND McELENEr, W. J.~{1941) Ibid., 1, 737.

Buens, E. L. and Schenken, J. R. (1943) Cancer Res., 3, 691.

Clyde, J. D., Law, L. W. and Dunn, T. B.— (1959) J. rw/. Cancer Inst., 23, 717.
Dunn, T. B. — (1953) Ibid., 14, 1281. — (1959) in ‘ Bhrsiopatbology of Cancer ’, edited
by Horaburger, F. and Fisliman, W. H. London (Cnssei and Co. Ltd.), 2nd
edition, p. 38.

Fodlds, L. — (1956) .7. riaf. Cancer Inst., 17, 701.

Heap, M. a. and Laird, H. M. — (1956) Rep. Brit. Emp. Cancer Campqn, 34, 282.
Heston, W. E.— (1958) Ann. N. Y. Acad. Sci., 71, 931.

Idem AND Deeingeb, M. K.— (1952) J. nat. Cancer Inst., 13, lG7.~{lfl53) Proc. Sn-.

exp. Biol., K.Y., 82, 731.
lidem and Dunn, T. B. — (1956) Ibid., 18, 1309.
lidem and Le\tllian, W. D. (1950) Ibid., 10, 1139.

Hummel, K. — (1958) Anat. Rec., 132, 281.

Jones, E. E. — (1940) Amer. J. Cancer, 39, 94.

Muhlbock, 0. — (1950) J. nat. Cancer Inst., 10, 1259.

Peehn, R. T., DLon, j. M. and Schneideesian, M — (1954) Ibid., 14, S95.
PuLLiNGER, B. D. — (1952a) Brit. J. Cancer, 6, 09. — (19525) Ibid., 6, 78.— (1955) Ihid.,
9, 613.— (1959) Ibid., 13, 99.

Pybus, F. C. and JVIiiLEB, E. M. — (1940) A»«er. J. Cancer, 40, 47.

Shimbin, M. B. — (1945) in a Sj^mposium on Mammary Tumors in Jlice. Atncr.

Advanc. Sci., Wash., p. 85.

SmoNDS, J. P. (1925) J. Cancer Res., 9, 329.

VAN Ryssel, T. G. and Muhlbock, 0.— (1955) J. nat. Cancer Inst.. 16, C59.
WHJTEltEAD, R. — (1932) J. Path. Bact., 35, 415.

Woolley, G. and Little, C. C.— (1945) Cancer Res., 5, 193

Addend tnvr.

Thanks to the kind collaboration of Professor JI. G. P. St oker
of the M.R.G. Virology Unit, Glasgow, sample sera "Ano

been tested for polyoma antibody. Hjemagglutmation inhibition titrcs oiei 1/3-

found in both strains.

TFSTS FOK :MAM^1AKV TUMOl'U

CJ-I, AND Dill, MDl'SK STDAIN^

B B. ITLLIXCKU

f,.om ,he Cancer Ecscarch Dc,>a,1,nrnL Eo,,al Era,.,,, Men. rial W.,Ual C!,.,rr
Rpocivi'd for inihlicAtiix' Kclininry 2!V

IlEFERE>-CE data for eventual comparison of jj'*'
mammarj’ carcinoma in C,H,;Pu female m.e(. and . I

r^-ith that to be seen after substitution of graded amounts of . ^ nan J ';
hormones have been recorded. Before these snhsl.tni.ons eouid ' " ’ ' ^

it was also necessary to examine this stram for i.resenee or ab^em'e of t In jnammar
tumour agent discovered by Bittner. Xo evidence of tnfeelion n itli n' ag.ml na .
found among the forebears by Heston and Ins mdleagnes (llf'^lnn. I • .'S) nor Inn.
any been detected in this laboratory. Evidence has hmm sought for ni-l)n-,)iii::
records, from injections of spleen and tnnnnir extracts, by attempt-' at lori!/
activation of the pathogenic action of the agent and by cross.s\»elvling nen - >oin
RIIIj mice on C3H, nursing mothci's.

MATEUIAES AND Mf.TIlon.s

The source, management and methods of cxnmii\ation of the the two stiMiim
have been described {Pullinger and Iverson. HKltt).

Detection of the. mammary tumour aejent

If the agent is present in small amount the infection may not be revealed by
maramarj' carcinoma in one generation of mothens but only in bia-eders of suc-
ceeding generations (Andervont. Itt.oO). Thus the most reliable biologie;d criterion
for judging absence of agent is considered to be fnilure to find an accumulation
ofmammarj' carcinomas in sublines in breeders observed through several genera-
tions (Heston, 1958). When the agent is present in greater amounts, whether by
natural infection or by injection of extracts of tissues or tumours, its pathological
action is readily revealed in susceptible agent-free females after breeding. No
0 jective measure of the amount of agent is available. The origin and ])edigree
records of the mice used for testing must be free of suspicion that they themselves
are carrjnng this agent. The Hlll^ strain has been used for this prinmse, hence the
need to examine recent breeding records.

force-breeding test was introduced (Bagg and
breed th’pmml^^ considered necessary- by some workers cither to force-

ousnormaTSr.r stagnation, or, by others, to allow numcr-

favourable for conditions are believed to be the most

BWO) nor Bittne? agZ carcmoina. Neither Little and Pearson

breeding. A direct r^ltil m ^ favourable influence of force-

2S0

B. D. B-DLLlJrSEE

sS£SS-m-H“S3

Bittner rnammarj^ tumour agent. Nor has any attempt been macle‘to iLrca=e
the amount of this agent by culture in cells m vifro in these laboratories Xn
account of such studies elsewhere has been found. It is not known whether thi.
branch of the original C3H strain carries the polyoma agent. No spontaneoin

bv Plvd^T T "no ''^Sent and described

by Clyde Law and Dunn (1959) or by Stanton e( at. (1959) hare been obsencd
m this colony nor in any of the experimental mice.

The natural occurrence and forced production of adenomas (h)-pcrpkstic
nodules) to detect the mammary tumour agent in young mice u'ere iin'cstigated.
To avoid repetition these observations and experiments are recorded with their
results.

RESULTS

ItllJf breeding records and mammary carcinoma

An incidence of this tumour of 12 in 482 breeders or 2-5 per cent in the iir.’t
34 generations has been recorded (Pullinger, 1955). Among 544 breeders in
generations F.35 to F.52 it w'as 14 or 2-6 per cent (Pullinger and Iverson, KWh).
In the F.35 generation 6 mammary adenocarcinomas arose in 6 out of 57 breeders
thus giving a higher incidence than had ever been found before. Tliis rise nu'glit
have been due to increase in a persisting small amount of agent or to its more
recent acquisition. The attempt was made to breed from tumour-bearing females
in F.35 to look for tumour increase in sublines. As mammary carcinoma arose so
late in life at an average age of 19-6 months in these G females, it was by chance
that 1 subline, derii'^ed by direct descent, and 2 sublines, derived from sililings
of other tumour bearers, had been bred when the unusiiai liigh incidence was
noted. All 6 tumour-hearing females belonged to different sublines in F.3r>, having a
common ancestor far back in F.24. Neither this common ancestor nor atu' inter-
vening progenitors had developed overt mammary carcinoma but the mother in
F.34 of one of the 6 tumour bearers in F.35 was found at necropsy and hy hiilk-
staining to have a microscopic adenocarcinoma of Type A (purely acinar) in a
first left nipple area. Her daughter developed an anaplastic carcinoma in t le
second right nipple region at 16 months of age. A biopsy was done and t ir«
and a half months later the prima^' had recurred. At necropsy mas.sivo piiltnonaia-
metastases were found. Subline I, directly descended from thi.s tumour-bcarmg
female, was bred through 5 generations which included 39 effective breeder- f iat
lived to the ages given in Table I. None developed (B tm

two sublines derived from siblings of 2 of the other

tumour-bearing fenialt"

one, subline VI, was bred for 9 generations and included 3G effective '"-cederr r
iven in Table I. No mammary tumour aro.se m tlic.se da-ccmlam. •

lived to the ages giv. ...

The third subline was bred for 2 generations witli

effective breeders, ^oitc

developed this tumour. In assessing incidence, although ^

mated females, only those that had more than -
over have been considered effective. None that lailed '

developed mammary carcinoma. From the Table I o u 1 ’o

ublincs I nnd VI

tests EOE jMAJDIARY tumour agent in C 3 H/

and RIH/ MOUSE STRAINS 2Sl

282

B. D. PULLINGEE

(1960) it can be seen that the unusually high incidence found in E .
maintained m succeeding generations whether breeding was restricfar'orMm
The survival ages m most later generations was not less than in F 35 Since tlic

of 23 and 23 out of 25 breeders in F.53 and F.54 have died without (unioan
The remaining 5 are 23 months of age or over and are free of tumours. Tlic inh.ancc
just recorded is the only one among a total of 26 mammary tumour bcnrm in
5^ generations in which a mother and daughter were found with tliesc tumours
one microscopic and one overt. Tlie great-grandmother of one female in F17
with a mammarj^ carcinoma also bore this tjTie of tumour. The only .sil.lin.vs
round with overt mammary carcinoma were 2 out of 4 in F.49. Once previously
in P.13, one sister developed a macroscopic and one a microscopic carcinoma. Xn
other close relationships of tumour-bearing females were found.

Strain

Presumptive tests for mammary tumour agent

While pedigree records were accumulating, some presumptive tests for agent
were done.

1. Extracts of spleen derived from surplus CgH/ males in early litters were made
by spimiing a 10 per cent suspension of homogenised tissues for 15 minutc.-f at
14,000 g. Intraperitoneal injections of 0-05 ml. of supernatant fluid were made
into each of 13 Rllly females aged 3 to 4 weeks. All were subsequently force-I)red.
Nine in the P.l generation and 3 in P.2 survived from 8 to 21 months after hearing
3 or more litters. No mammar}' carcinoma arose (Table I).

2. An extract was later prepared from a mamniarj' carcinoma iu a (yiy
breeder. Intraperitoneal injection of 0-5 ml. of supernatant fluid from a 10 per
cent suspension spun at 14,000 g. were made into 9 Fill; females aged .'1 to 4
weeks. All were subsequently force-bred. Six survived for 8 to 24 months after
bearing 3 or more litters. No mammary carcinoma arose (Table I).

3. Tv'enty-nine RIII^ females were bom in the presence of, and were Jeh
with 15 G3H, nursing mothers. Their onm mothers were removed within, at mod.
16 hours of birth. These P.l cross-suckled RIII^ females and 6 of their 1 .2 jirogem
were subsequently force-bred. Nineteen of the original 2!) bore 3 or more litter'
and lived for 8 to 26 months. One breeder only of the 1 9 in F. 1 devclojied a iieriniia^
mammary carcinoma of compound organoid type at 9 months of age after lieanng
6 litters. Neither of her two C3H/ foster mothers developed mammary earcinonm.
Six only of the pooled F.2 generation were reared and mated. They lived to the
acres given in Table I, bore multiple litters, and none developed mammary carci-

noma. , , ,

4. It was previously reported that adenomatous mammnry nodu
found regularly in the mammae of young females under a year old ' '
strains infected with milk-factor, with the exception of Strain A. hut '
was seen in young virgins or breeders under this age after ori'din!

of agent (Pullinger, 1952, 1955). Precise figures for thc.se
C,H (Andenmnt) strain infected with agent are not knov n. Soduk^
to tio C,H, substratos fread from agent in female., of 15
data for younger mice were not given (Jones, 19:>1). Thu.s no J’' .•
made. Nevertheless a systematic examination of inilk-staincci prej a .

tests EOE >UM>.AET TU»,0«E AC.BKT .K C 3 U, AS,. MOUSE STEMSS

5-0 nipple areas Of 5- O.H,virgi,ao,Ealosnp<l „ mnntl.s rvas nn.lertaUen. T

h5T,_er^asticnodde|nere^^^^^^^ , |„e patl.osenie

ssssssess

or oestradiol but that, using the same technique, none w.as found m imco of tli
same strain that had been freed of agent. Twelve C^H, fcnialc.s "''"’’''''’‘"''''J ‘
at 2 months of age were given 2 doses of 400 //g. oestradiol in acetone />rr ru n,
at an interval of 20 days. At S months of age the 1 ] survivors nere killed and all
ninple areas bulk-stained and examined. Regression of physiological resjionse
had occurred in all and no carcinoma or adenoma was found. \\ hen a
test was done on 10 G3H females (derived from another subhiic) infected with
agent, adenomas were found in aU 10 (Pullinger, 1047). Similarly 4 RlII/
injected with spleen extracts and 13 males nursed with their sisters by O3H,
mothers were castrated, treated with 3 doses of 400 /(g. ocstrone and 1 dose of
400 /ig. progesterone, and were killed at 8 months of age and all fat jiads examined
by bulk-staining. No adenoma or carcinoma was found. Tn previous similar
tests of Rin males carrying mammary tumour agent, adenomas were found in
28 and carcinoma in 9 out of 30 mice but in none out of 1.S RIIIj. males (without
agent) similarly treated (PuUinger, 1947). The susceptibility of the RlII/ mice
to agent was again tested intraperitoneally with CB.d tumour extract. Ten out.
of 13 R.III^ sucklings developed mammary carcinoma after they had been force-
bred (Table I). This CBA subline had previously been found to carry the agent
(PuUinger, 1953).

CgH f breeding records and, mammary carcinoma

The overall incidence was 25-9 per cent (PuUinger and Iversen, 1900), These
tumours were distributed haphazard in sublines. Records including 3 generations
of ancestors of 26 out of 28 of the tumour-bearing females were available for
pedigree analysis. The records of the great-grandmothers of 2 were unknown
owing to transfer of the original breeding litter to this laboratorj% but neither
their mothers nor grandmothers had mammary carcinoma. None of the 20
remaining tumour bearers had 3 ancestors with these tumours in the direct line
of descent. Five pairs of mothers and daughters had tumours of this kind and one
of the latter had also a grandmother with mammary carcinoma. The only other

"vf r f n ^ great-grandmothers and 4 with grand-

mothers which had borne these tumours. The remaining 9 (about a thirdl had
no knowm progemtor with this carcinoma. These data reveal no evidence of
accumulation of mammary carcinoma in sublines.

C03IMENT

Mil,

tion of mammaiy^ carcinomas in any sublines such as is

inammarj. tumour agent is present MtiaZ .fl ■ n ^ the

tests for presumptive evideCol rageT'iTm- "T''
arose m 43 RIH^ test females that had bfen W Serf T carcinoma

plastic) nodules were found in 570 nipple rLoS S

giuns 01 0/ virgin females aged

284

B. B. PULLINGER

11 months These two observations, namely 1 mammai^^ carcinoma in a vo.m<^
cross-sucked breeder and 2 nodules m smung virgin females, provide tlie onlv
evidence that could be construed as indicating that the mammarj' tumour
was res^nsible for all or part of the mammarj’- carcinoma incidence in the C H
strain. The single adenocarcinoma in 43 RIII^ test mice could have arisen snoin
taneously ” independently of the experimental procedures. However, it arnse
earlier, at 281 days, than any previously found in RIII^ breeders. Thc'raiwe for
the first 12 carcinomas in F.l to F.34 was 389 to 753 days. In the present genera-
tions, F.35 to F.52, two of 14 adenocarcinomas were found at 327 and 330 davs
of age. Thus with larger numbers of tumour-bearing mice a wider spread in age
incidence became evident. Tliis seems a more likely e.xplanation of the earlier
occurrence of this single carcinoma than that one only out of 43 RIII/ test mice
was infected by an agent derived from the CgH/ strain.

The value of the obseramtion of 2 adenomatous nodules at 1 1 months of age
in 570 nipple regions cannot be assessed without further data about the earliest
age when they appear in this strain. In the RIII^ mice none has been scon before
one year of age (Pullinger, 1952, 1955).

SUjMHIAEY

1 . Pedigree records of 11 generations of C 3 H/ and of the last IS generations of
RIII^ breeding females have failed to provide evidence of an accumulation of
mammary carcinoma on sublines.

2. No evidence of the mammary tumour agent of Bittner was found in testa
with injected spleen or tumour extracts nor any indisputable evidence among
susceptible agent-free cross-suckled young mice.

3 . Experiments designed to stimulate the pathogenic activity of tlic mammary
tumour agent failed to reveal evidence of its presence.

REFERENCES

AnnEBVONT, H. B.— (1950) J. nat. Cancer Inst., 11, 545.

Bagg, H. J. Aim Jacksen, J. — (1937) Amer. J. Cancer, 30, 539.

Bittner, J. J. — (1948) Cancer Res., 8, 625. / , 09 -it

Clyde, J. D., Law, L. W. and Dunn, T. B.-(1959) J. nal. Cancer Inst., 23. /!-.
Heston, W. E.— (1958) Ann. N.Y. Acad. Set., 71, 931.

Jones, E. E.— (1951) Cancer Res., 11, 260.

Little, C. C. and Pearson, J. — (1940) Amer. J. Cancer, 38, _-4.

Muhlbock, 0. — (1950) J. nari Ca?icer/««L, 10, 1259. r _ncvM /61V/ t 3 .

Idem, VAN Ebbenhobst Tbngbehgen, W. and van Russel, Th. G. (1. •>-)

Pm^GER, B. D.— (1947) Brit. J. Cancer, 1, 177.— (1952) lint., 6, 69.— (19o3) RaL
7, 490.— (1955) Ibid., 9, 613.

Idem AND IVEESEN, S. — (1960) 14, R ff flW!)) xnl.

Stanton, M. F., Steivaet, S. E., Eddy, B. E. and BurnKUEU., R. R. (i- M

Cancer Inst., 23, 1441.

Addendum (24.5.60),

Thanks to the kind collaboration of Professor M. G- R ,ni( o h.of

of the M.R.C. Virologj' Unit, Glasgow, sample sera fro ... ovf/l/32t> ttcw

been tested for polyoma antibody. Hmmagglutmat.on mh.b.t.on titrc )/

found in both strains.

expeeiences with HYPOPHYRKCTOHY IX MK'l-..

Ckwekia of CoMrEF.TE 1 H:>^ovae

STRETTOK YOU^'G A>-1> LUCY E. I'EASKU

From tU CUnko-pathohgkal Lahorntork>< of Cnnrrr / r-^mrc .

London, It .C.J;

Ecceived for pubVicntion FoEnmn- 3. IttCiO

It is important to users of InTopPyscctomised tnice to know if rcnovnl of tin-
pituitarj^ gknd has been complete. Infommtton on '""f, .'Jj'",!,,

by posl-mortem examination of each pttmtary fossti. earned < nt ^ h r ^ hr.
inspection using a relatively low power micro.scope .'Jf

cutLg serial histological sections of the entire area after decalcirieatinn and
paraffe embedding. The results of examining 432 mice by the latter method

have already been reported (Young, 1959). _

Although the histological methods described permit the recognition ot very
small fragments, they are both laborious and time-consuming and it would be of
great benefit if other criteria, more readilj’ available, could be shown to give
equally useful results. Indirect evidence may be obtained by observing and
measuring the effects of hormone ivithdrawal on the appropriate target organs.
Thus, somatic growth, if still occurring at the time of complete removal, will
cease thereafter, and the mammary glands, gonads, adrenals and other target
organs will undergo progressive atrophy. The response of the mammary glands is
of particular interest in this laboratory, for the Mammogenic Growth Response
in hypophysectomised mice has been used in investigations into the occurrence of
growth promoting substances in normal female urine.

Griffiths (1941) has used the criteria of “ cessation of growth, extreme atrophy
of adrenals and testes and absence of macroscopic pituitarj’ fragments ”.
Lostroh and Jordan (1955) examined the adrenals, ovaries, and uteri both
macroscopically and by weighing. The pituitaiy^ fossa was examined with n
“ binocular microscope ” but sections were not cut. Bahner and von Graff
(1957), after cutting serial sections of 25 mouse heads, considered that “ rem-
nants of the hjTDophysis are rather reliably indicated by gonadal weight ” ; thev
particularly favoured seminal vesicle weight.

It IS the puipose of this paper to compare several of these criteria with the
histological findings after cutting serial sections of over 200 mouse heads and to

St thif W °ff^\-tisfactory alternative to the latter. We

beher e that a detailed comparison of this nature has not previously been reported.

j. t-i 1.^ 1^ » 1

tcdmiJii’desSibS^y Thil?”''!"®

^ . or as .easy as AfJr opera^r^

2S6

STBETTON YOVNG ANV LUCY E. ERASER

oats and water ad libitum. No glucose or antibiotic was given. The mice were

weighed daily for 14 days and at progressively longer intervals tiicrcaftcr if
allowed to survive.

/rr ™ hilling, fi.ving and staining pelts has been dcscriliwl

(Hadneld and Young, 1956). The stained and mounted glands were examined
for the presence of “ clubs ”, rounded, denselj’- staining structures found at the
ends of groxving ducts and having a diameter more than tuice that of the duct
from which they arise. They consisted of closely-packed epithelial cells, many of
which were in mitosis. Enumeration of clubs therefore gave an estimate of the
degree of mitotic activity and hence a measure of the rate at which new duct
formation was taldng place. The mean number of “ clubs ” per mouse wa<
regarded as the Mammogenic Growth Besponse (M.G.R.).

The heads of the mice xvere fixed, decalcified, trimmed, embedded and cut
serially. Besidual fragments of pars anterior xvere recorded as size -f if only a
few cells w'^ere found ; size if large enough for tj'pical histological structure
to be recognised, or size -f- -f- -f- if they amounted to a substantia! fraction of the
whole gland (Young, 1955). In view of the possibility^ that pituitary fragments
might be transplanted into the brain at operation, the pituitary fossae of 03 of the
mice were examined serially without remmdng the brain.

Table I. — Details of Treatment Given to 207 AjG Mice, Hypopliijsectomised at nfJ
between 22 and 27 Days whose Heads have been Examined Hislologknfhj for
Residual Pituitary Fragments

DnvH .'

Number
of mice
86
21 *
43
23
34

Nature of injection
None

Oestrone -f progesterone

Prolactin

Growth liormone

Dose : per mouse
per 5 days
Nil

1-25 pz- ■+ 1-5 mg.
50 i.u.

0-2 mg.-l-O mg.

Operation Death

7 142

8 If'

8 10

- Ifi

7 14

* In these 21 mice the operation was intenfionnlly incomplete.

Most of the mice had been used as controls in experiments on
tropliic activity of urines and hormones. The details of injections o a i
whie heads were cut histologically are given in Table I. After
carcasses of the 107 uninjected mice, the organs under investigation « ere ■ ■

out as follows : ...

Testes . • . in 69 animals

Seminal vesicles . „ OS „

Adrenals ...» 09 »

The tissues were lightly blotted on filter paper and xveighed
mg. torsion balances. A further 169 "

preparing Tables IV and Y. The heads of these were not cut .

BESULTS

Somatic weight

Since our mice xvere hypophysectomised
of the most noticeable effects of operation

ori'

while they were growing -

vas the cessation of growth.

CRITERU OF COMFLRTE IIYFOVHYMXTOMY

Blowiin Table II and Pig. ' "'“c™ I

ml'ltb™oTaU'a«ce amber of .nice have been Ml.nvrd f.T C.

Fig, 1. — Comparison of mean somatic wciglits of hj’popliyscctomisei)
and intact mice over 7 days.

Intact mice. Hypophyscctomiicd mice.

288

STEETTON YOUNG AND LUCY E, ERASER

days. It is to be noted that the mean weight of comnJptplv
mice ^d not return to its pre-operational lefe] although tlie weiStf

Taele II.— Cmapamon of Mean Somatic Weights of
Hypophysectomised and Intact Mice Over 7 Days

Hjpophysectomised

Intact mice

Days after

Number

Mean weight

Number

Mean we

operation

of mice

(g-)

of mice

(g-)

12-53

10-8

11-56

11-5

10-95

12-2

11-42

11-8

11-47

12-9

11-3S

12-7

11-53

13-7

11-58

14-5

Varying numbers in the second column are due to the fact that these mice belonged to pevorid
different groups and daily weighings were omitted on Sundays and public holidays.

To test wliether fragments of pars anterior secreted enough groivtli hormone to
influence weight, the individual body weights of 177 mice were examined. Tlie
mice were classified according to the histological size of the fragment found.
Thus, 124 were completely hypophysectomised ; 32 had fragments of size r :
while a further 21, intentionally incomplete removals, had fragments of size

The percentage gain or loss of weight 7 days after operation was comj)arcd
in these three groups and the results are given in Table III. From this it can I'c
seen that the mean loss of weight in completely hj'pophysectoniised mice was
greater than in those shown to possess size + fragments and tliis difference ::r
standard error was statistically significant (P < 0-05). In the case of mice nith
size + + + fragments there was a mean gain in weight instead of a loss and tlii'
difference 4- standard error was very highly significant (P < 0-001).

Table lll.—Data Eelating to Percentage Somatic Weight Gain or Loss 7 Pi;;<
After Operation between Grotips of Mice : (a) Compktely Hypophyscclomtsn .
(b) with Residual Fragments of Pars Anterior Size + ; (c) trtlh Iksv v>
Fragments of Pars Anterior Size + + -)-

Number of mice .

Mean % weight gain or loss in
7 days

Standard error of mean

Without

remnants

124

-G-19

0-432

With hi.stoIogipiiI "’ith lii-tolori' -'!
pituitary remnants ])ituitarv mnn.in '
(SW-f)

-1-95
1 - 732

-J. 3
2 -It'S

Mammary glands -^rcto-

The possibility of using the mammogenic growth
mised mice, treated noth oestrone and progesterone, as a y-

of “ mammotrophin ” b3’^ fragments of pars antenor nas

CRITERIA OF COMI’EETH HVrOFUYSECTOM V

poup“S i” jLlcd ulfc' lllilv‘« ilh r«.l.'.l i.m of F,.t,,rl in in
saline over the same period. Tlic total do.sc.s for oneh group ranpml from < mi'.
nTto 2 5 mg. per nlouse per a days. The auiumls were kd ed ami sk.nned on
tlfe si.xth day mid the mammogenic growl li response eslimaled. .'I

was repeated giving doses of growth hormone ranging from o-Oimn.l. mg. to ..

3.~Ilegre.ssion of mammarj- growth rc.sjfoiiPC on tioso of prolactin in
nypophysectomised mice treated with ocslronc and ifrogc^itcronc.

m^., instead of prolactin. The results are given in Tables IV and V and in
tnmi=pd^m- ’ r ®;PP® 2 .rs that the mammogenic groivth response in hypophvsec-
with variahlp ^oses of oestrone and progesterone together

locaritVim of P™l^ctin or growth hormone was proportional to the

g nthm of the doses of the latter, for in both cases linear regression was

PrSnJ? 1° of Mammogenic Groxulh Rcsponsc.H on

j <^rune 1 ng_ and 7-5 mg. per Mouse per 5 Days

Mammarj' growth
response

g./mouse/5 days) (Mean clubs/mouse) Number of mice

0*0008 in

S'F ■ •;

« : :

• 101-45 . jf

Linear regression is highly significant P < o-OOl.

25)0

STBETTON YOUNG AND LUCY E. ERASER

physectomiied treated oes JoS'ln? ’‘•T,'

PE::r

been excreted by the residual pituitary fragments.

Table V. — Da(a Bdating to Regression of Mammogenic Growth Responses on Pa"
of Growth Hormone in Hypophysectomised Mice Treated with Oeslrone nii'J
Progesterone, 1-25 pg. and 7-5 mg. per Mouse per 5 Days

Growth hormone Mammai^' growth
dose reqjonse

(mg./monse/S days) (Jlean clubs/mouse) Number of mice

0-000032 . 21-00 . 13

0-00016 . 20-61 . 13

0-0008 . 25-53 . 13

0-004 . 22-84 . 13

0-02 - 59-76 . 13

0-1 . 65-07 . 13

0-5 . 97-46 . 13

Linear regression is statistically significant P <0-01.

Forty-three hypophysectomised mice injected m’th oestrone and progc^ ffoi
in arachis oil were examined histologically for residual fragment.^ o I” ' • j.
The total dose of oestrone was 1-25 pg. and of progesterone /-a mg.,
this being given at each injection, night and morning, for <5 daj's. i me n
shown to have residual fragments of pars anterior, all of them ?,\ze -■

The mean mammogenic growth response ± standard error m ic- ,

6-78 ± 4-3 compared with 4-74 ± 1-401 in the remaining 34 which had "
strable pituitary fragments. The difference was not statiaticalb «

CRITERIA or COMPLETE nYl’OlMIYSECn'oM V

(P =: 0-1). Each of these groups could he divided \ip. liuwever. dependiii}' iiu
whether the first iujcctiou was given (> or less, or 7 or luon'. <lays after oper.Uioii.
In the latter case, the G mice having fragment, s of size -p had a mean mammtie,.|,jp
growth response of 2-o ± M.a:? while the rom.aining 22 with no denion^'l r.iLle
fragments had a moan mammogenic growth response of I-Rli ■ (i rejo. nii(’e
again the difference was not statistically signilicant (/’ > (t'2).

A comparison was also made between niiuumogenie growth re~pon>.ei. nf
complete/^ hj-pophysectomised mice {a) having their first inject ion G davs or le.-,
after operation (12 mice, mean M.G.R. = in-pg ,i„.jr

first injection 7 days or more after operation (22 mice mean ^I C R l.-i,-, ,
0'520L The minp hnvinrr n 1_ „ r.

iuiL'u. iiluilii niitl (6) liHvintr thr^ir

first injection 7 days or more after operation (22 mice, mean M.C.R, ,

0 520) The mice having a first injection a week or more after oiteratio-i gave
consistently low mammogenic growth responses, while those inje<-t.-.l within a
lew clajs ot operation not onlv cave Litrii

. . . ,, , o - tu illlMC lllHT njH'niTUi:i viwv

consistently low mammogenic growth responses, while those injeet.al within a

niammogenie growth respooM-. hut

two another. 'Phe .lilT.T.mee heln,-e„ the

groups nas verj- highly significant (P<()-i)(ii) R i< iKK‘,il)Ii.

the secretion of oestrogen of fragments of anterior pituitarv. Sine*-

been shown to be activelv nnmm ''”l''>r<‘ prolaetin has

trophin activity indkectlv hTmofnrV m'”’'-'’ H'-nado.

bjTophysectomied mice treated with L'rowlh response j,,

sectomisedniicetreaternS^ 'f'venty-lhree In-pophl"

histologically for evidence nf -f ' ° monso jier "> davs were mined

W in e'mice. Srimd^fme?' ‘>f size :

compared to 1-35 for the 17 mine • i" tFO"'*'! response of Mt;

that no measurable gonadot^^^^^^^^ no fragments" were fomi 1 . R I , ,

“-nithfragmentsSze by this metliod io

operation the^pT available^for stlidv A?^f *^PP'’npri!ito target

tbe42 animrf^ ‘’-W-s nhov

Jrd error of had a memTteTth w^”u

of 1-84 nig. j 0-08a^il,~ ~ "^th a mean seminal vpcIpI ■ ''^’S^t .slan-
"■eight of 39-32 m 's aoa ^ + fragments •'ininials)

!ng- ± 0-249 mg ^ ™g- ^oth a mean seSS ? test i.s

'"complete opefationsM '^th size 4-4-+ i., "'^''ght of 2-27

" "'^"'i seminal vlS e wv ^'oight of^rt 92 (intentionally

4i- -"t-vt+v =.4~ 4 ■■ * "

s ’ ^non the value of

292

STRETTON YOUNG AND LUCY E. FRASER

P < 0;1 cou d be halved to P < 0-05, which is statistically significant In n,!
with size + + + fragments both testis and seminal vesicle weWits showed 1!^
ences which were highly significant {P < 0-001 in both cases) ” ^ ^ ^

Adrenal iveighfs

Just as deprivation of gonadotrophin gives rise to atrophy of testes ami semim!
vesicles, so deprivation of adrenocorticotrophic hormone leads to atropiiv of the
atonals. As in the examination of testis weights, 69 mice were available for the
adrenal study. Porty-two completely hj^ioph^-sectomised animals had a mc.in
nmight ± standard error of M7 mg. ± 0-022 mg. ; 6 mice with size + framiienL
had a mean adrenal weight of 1-22 mg. ± 0-054 mg. ; while the 21 mice with
+ + + fragments had a mean adrenal weight of 2-30 mg. d: 0-0!)0 mg. Xn
significant difference in mean adrenal weights was found between mice possc.^-dne
size -f- fragments and those with none (P > 0-2) ; however, there was a liiglilv
significant difference in the comparison of mice with size +++ fragments mill
those containing none (P < 0-001).

DISCUSSION

From a consideration of these results it is clear that the presence or abscticc
of size -f + -j- fragments was associated ndth verj' significant differences in somatic
weight gain or loss, and also with significant differences in the iveights of teste?,
seminal vesicles and adrenals. Unless they were left behind deliberately, frag-
ments of size + + -h were very uncommon. Their identification was, therefore,
relatively unimportant but their presence was detected by daily weighings of the
operated mice, since animals gaining weight regular^ after the first few post-
operative days have been found to possess fragments of this size (Fig. 5).

The discovery of mice with size -f- fragments was a more important problem,
for these occurred more frequently. It appears that significant difference.? in
somatic and seminal vesicle weights were present between mice containing .size ■*-
fragments and those with none. The seminal vesicle weight difference was onm^
a satisfactory indicator by Bahner and von Graff (1957) but it has tiie disadi nn ,ig'
that it entails either another operation, or death of the mouse. In flie appru ’
of mice for experiment the advantages lie clearly with selection by soma ic kcis > ■
Unfortunately, the 156 mice we have studied in this connection, verc oo ea
number to permit an accurate forecast of the level of weight increase o
fidently associated with the presence of size -j- fragments. It las
habit, however, to discard as unsuitable any mouse exceeding its opera
by 10 per cent on the seventh post-operative day.

Size + fragments appear to have been insufficiently'^ active ‘

a significantly increased mammogenic growth response with iiqec ion ,
and progesterone and this has apparently also been triie of mjec ion ^
given alone. Since fragments of size + are virtually the only' size ^
in our mice, this implies that it is no longer necessary' for us o ci " ^

of pituitaiy fossae from hypophy'sectomised mice trea e
progesterone, and used in the determination of mammogenic gr .-ippo.ui''-

Occasionally' no pituitary' fragments were found in a mouse ,.,,1-.

biologically to show evidence of pituitary activity' as ju g .
following the operation, or a higher mammary growth respons

CRITERIA or COMPLETE HYPOPH YSIRTOM Y

following oestrone + progesterone ndminislnition. 'I’liere an* sc'Veral po; .jlili'
explanations :

1. The mouse might liavc been uiuleriiourislied or partly dehv<lr.i1«il
at the time of operation and gained weiglit s>disi‘(iiiently to a normal levi-l,

2. Fragments of pars anterior might have lieen left la'himl wliieli
continued to secrete liormones for a sliort time Inil did not survive per-
manently. Such fragments liave. in fact, been oeeasion.allv oliw-tved
(Young, lOotl).

have btn’lLnspkSed^S’t^^^^ fragments of pars anterior n.igl.f

of S,t S “"W "O longeTb/dlre r

294

STEETTON YOX7NG AND LUCY E. FRASER

It is interesting to note that there was no evidence of regeneration of size -i-
ragments up to three months after the operation, and bony regeneration of tlie
removed plate of basi-occiput was incomplete.

SUMMARY

1. Over 200 hj^iophysectomised mice were examined histologically for tlic
presence of residual fragments of pituitary. The presence or absence of pars
anterior fragments was compared statistically ■noth differences in somatic weight ;
testis weight ; seminal vesicle weight and adrenal w’eight, and with the mamino-
genic grovdih response after treatment with oestrone and progesterone.

2. The presence or absence of size + fragments was associated with significant
differences in somatic and seminal vesicle weights. Differences in testis weight
and adrenal w^eight were not statistically significant.

3. It Avas not found possible to forecast accurate!}' the presence of size -t
pars anterior fragments from a consideration of w'eight alone.

4. Jlice w'ith size + pars anterior fragments did not show significantly
increased mammogenic growth responses ivhen treated with oestrone I-25 /;g.
and progesterone 7-5 mg. per mouse per 5 days.

5. In mice completely h}'pophysectonused and treated with oestrone

and progesterone 7-5 mg. per mouse per 5 days the mammogenic growth re-sjion^c
was significantly greater if injections were started less than 6 days after operation
than if they were started 7 days or more after operation.

IVe are indebted to Dr. C. C. Spicer, Head of the Division of Statistics of the
Imperial Cancer Research Pund, for adr'ice in statistical methods, and to Mc-'W.--.

J. Gilbert, T. O’Connor and P. V. Sharp for technical assistance.

We are grateful also to the Endocrinology Study Section of the hatmaal
Institutes of Health, Bethesda, Ma^land, U.S.A. for the gifts of growth horinoac
and prolactin, which have made tliis work possible.

REFERENCES

Bahnee, F. and von Graff, H.— (1957) Acfa endocr., Copenhagen, 24, 3,13.
Griffiths, M. — (1941) J. Physiol., 100, 104.

Hadfield, G. and Young, S.-(1956) Brif. J Cancer, 10, 324.

Losteoh, a. j. and Jordan, C. W.-(1955) Pros. Soc. exp. Biol. J .1 .. -"<•

Thomas, F.— (1938) Endocrinology, 23, 99.

Young, S.— (1959) Brit. J. Cancer, 13, 208.

CASCKOC-BJv-S BORMED IX v'l' i' " '

FOmUTm-or3.4-BBXZORYUEXEHi-<M MAR' II A1

W. DAVIES .oi> -1. H. WIEMSHI EM

riiii'^r.'ily "/ ' 'I ’ ■ .1 I ' ’

From tht Depnrhncni oj Chantstrtj

Bcccivod for Mnnli I •. I

.1.A

Mii-Un'r %M'U V.ut'wn
for cMumt'.i’ Ev
njl in <■11.. El r

tlu' wM>t oEtniiii'iI in Dx’ ''

i><-

.,n./

i'llH-.

r..

•i'!

'rHE present investigation is to (Ictermine
carcinogens are formed in normal domestic sU nations,
of foods under conditions approximating to those ap]dying
Though the 3,4-benzop>'rene (BP) found m the .«oot oht an
of foods and also in the smoked food itself anses from the
the smoke (Tilgner and Vlnller, Itiru ; Dohe.s, llopE ami Mila 1 .m , ; 1. u!.

Dungal, 1958), in some cases the foodstuff itself can. nhen heate.! '’''■r,,Vr '
carcinogens. Thus nine polvcyclic aromatic hvdroraThon- imdmlnv,: El U<.a'-
been detected in coffee soots, though they were not pre.-ent in tin- U-an-

before heating (Kuratsune and Huepcr, ItbaS), Moreover in the " ehar fonu-l
in making “cakes” (fJ.S.A.) BP is dctcctcrl by a speetrophotonn-trie imth-d
which as used by Kuratsune (195G) is considered completely reliable (Pie.-er. 1 'X>~ j
Another group of polycyclic hydrocarbons containing BP i« tue^.ent in tobat-ro
smoke though not in the original tobacco (Kennaway ami liindsey, I'.i.'ts), With
regard to the temperature attained, it is estimated fKiiratsiim. and llne^HT,
1958, p. 38) that the coffee beans on roasting may be exjiost'd to a teinpeniinn’
of 540° C., and it seems likety that the wood and tobacco an* expo-.-d to even
higher temperatures. These materials, and most foodstuffs which are Imaled,
contain polysaccharides ; hence it was decided to examine the products obtuimHl
by heating starch, which is present in flourj*- mixtures which an* sometime-,
strongly heated.

A temperature of 370-390° C. was chosen as this is likclv t<i he reached in
some cooking operations, e.g. we have found that the surface temperature of
toasting bread may reach 390-400° G., for this is the temperature rapidly attained
when a mica sheathed thermocouple is placed in the same po.sition ns bread in nn
electric toaster. It may be that some temperatures in cooking are even higher,

^ considers that fats in contact with hot metal may rcacli tOO-OOtf
E. Ihis IS presumably the temperature range used bv Sarasin (15)1 HI who
destructively distilled starch (apparently with a flame), and wiio claiincd toluene
phenols, furans, ketones, fatty acids, and a compound GiaHi-O ; however most

Bie aqueous (bstfflate formed at ordinavu uressupo eout»i„„rr “ S

..r, partner u-fiC, .vas aW sOub.e; the Neutral p^raL^re^Slr '

296

W. DA■^^ES ANT) J. E. WILJrSHUEST

separations gave a crystalline complex with benzotrifuroxan (Bvhv md r

1958) which IS a reagent for aromatic t 5 mes. The aoueoiis diiiiUf ^ T’
contained no BP. aqueous distillate apparently

The carbonaceous residue in the pot contained much oxwgen (Found - 0 -

extract different samples) and the methvlene chloride

rrel nni ^''^shing With sodiuni hydroxide was chromatographed on <iiics
alumma to give a mixture of compounds from which on furtlicr
Jlw5 ® material noth the fluorescence spectrum of Bl> mv
obtained. The large amount of interfering material present precluded the use
of spectrophotometnc methods. The results we have obtained bv the fluorescence
method are, however, quite unequivocal.

EXPEBOIEXTAL

3 : 4-Bmzopijrene detection (BP).~A fluorescence method was used. The
exciting Taxation ivas essentially 365 m/i, a 4 mm. Chance OXl filter being n?cd
in conjunction with a mercury’- vapour lamp. A quartz condenser Jens was used
to illuminate the cell wliich was placed near the slit of a Hilger medium speclro-
graph. A glass train was used in the spectrograph so as to make use of tlie greater
dispersion of glass as compared arith quartz at 400 nyc, some three times greater
wdth this instrument ; exposure times were hov-ever considerably increased.
The cells avere U-tubes of Pyu-ex glass, one arm being 7 mm. and the other O-.'i
mm. diameter ; B7 sockets alloaa-ed each arm to be stoppered.

It aa'as necessary to deoxygenate the solution before ina'estigating tlie fluore-
scence spectrum ; this avas done by passing, for ten to fifteen minutes, a slow
stream of purified hydrogen through the solution. 0-01 /(g-/ml. of BP could be
readily and positively identified (in the absence of interfering materials
pg./ml.). _ , , ,

Chemicals. — ^All organic solamnts, chromatograpliic adsorbents and the aqueous
sodium hydroxide used were found to be free from BP. The isobexane ayns a
purified petroleum fraction b.p. 58-62° C. The luethydene chloride mentioned
in the detailed description of tlie experimental procedure has sometimc.s been
replaced by benzene. The commercial avheat starch used contained small
quantities of sodium chloride, lipids, and traces of protein.

The pyrolysis. — The retort aims a steel autoclave vessel, volume about 4 litres
and internal diameter 14 cm., of aadiich the head had been replaced by an iron
head equipped aadth a Pyrex condenser. To the retort heated by an ■

heated metal bath at 370-390° (bath temperature) the poavdered starch .m g-)
aa’as added all at once, the distillation head at once replaced, and the heau ^
continued for 25-30 minutes by aadiich time the distillation had almos ce. -
Thermocouples placed in the starch shoaved an internal temperature f '
than that of the bath. The retort aa-as rapidly cooled to J coil

the contend
worked up

temperature (15-20 minutes) by circulating avater through a staini
in the metal bath, and the retort then opened. If opened aboa-o 1 0 O.

The distillate (D), largely aqueous, ava.s

can spontaneously’^ ignite.

procedure.— The residue (amrying from 98
from the retort, aadiich avas tlien extracted for an hour aaith methylene ^ _
under reflux, cleaned, and used for a fresh operation.

The rcsidiic-s from tour

CARCIXOGKXS FORMRl) IX THK IIKATIXG (IF FOOD STCFFS

experiments were coarsely powdered, and caoh extraeled (Soxhlel) with ahoiil
500 ml. of methylene chlon’de until removal of (Inon*,seenee eea,>^('d. 'I'lu’.'ie four
extracts togetlier with the extracts from the retort, were (’oml)ined. ('xtraeted
thrice with 3 per cent aqncons sodium hydroxide, then with water and then dried
(MgSOj). The methylene chloride was di.stilled ofi’ through a short colnmn
packed v,ith helices, and gave a rc'sidne {about -t g.) of a light brown oil whose
solution in cyclohexane, together with cyclohexane washings of the beliees. w/is
clnomatographed on activated silica gel. The elnate. though verv (lnore.M’(>nt .
gave no fluorescent bands on further chromntogra])hv. the adsorb’ent beia.ming
uniformly fluorescent, so much .so that the band (>xpe(d('d bv the addition of even
miUi^ams of BP was completely ma.sked. 'Phis dinieid'ty was overetnue bv
simuftaneously using two columns which were identical exempt that one {\) eoii-

material from the carbonised starch, and the other
A as The zone m (B) was a guide in collecting the III* fraction from

fnnnrl • f ^ followed 111 all cbromatograpliic .separations h w.m
found convement to collect the elnate in relatively small e.p.a fra - , .s’ (n „ d T

chromatoLnhv nn -D *77^’ (tiycdohexane). but- repeale.l

interfering fluoLscenf '^''Parate the BP from the

(isohexane sLrated witli^d n^^^ '"V i ’ -Vn'- '’.vlformamidV

with those fractions^ d'niethylfonnamide). This ])art it inning ivas impeated

"f tile 4iiSatro^"tOT~5°i ",‘f 'sMmntioii the inclhylcno chloride cxlrnct
'Miducearact. ’ PI™'!’"'' ”•»» "orked e.xnctly n, will, H.e

extract ms aSnade''?' ifk a°e''''"l''“' Present in the methylene ohloricle
f»«nd to contain a mix ^.o .alkali solnhle materia

H, I2-7p creeirr H S' '"yP- 0. ronml ' 0 75 :

■"hich thus appears +n ’ ^^B^ires C, 75’.5 per cent H P’wTirtr-

presumably derived fromTe^- Z • t“earic achls

detected and tn. . ^^P'^s present in the stnrfxi, m

In additioiT. in only very smah ® ’ '^'"Sree

Pound ; C 5^7 '’’Tstalline benzo-trifuroxan deXaZ (Schiffs

After H. 3-3 per cent N i- 7 ^ ^™-P- ^80-1° C

'^•’^traction of 47? complex (by sol’utS’n i^rP obtained!

(Found: C 88-q material into isohexanef a formamide and

88 9^7"'^ S' P^^ een^TfeZuI J'oS “’stained

298

W. DAVIES AJSTD J. R. WILMSHUEST

oil f extracted from the aqueous disthlate by raethvlwio

chloride have not yet been examined.

^ estimation.— A series of BP solutions containing the same amount of
interfering material as the unknonm was used as a standard. An estimate of tlie
concentration of mterfermg material was obtained by u.r. absorption spectro-
photometry, by weiglit, and by fluorescence spectrophotometry. The imkiwwii
and standard solutions were photographed on the same plate ; comparison iras
by means of a microphotometer. Similar results were obtained when pure 111’
solutions were used as standards, in this case a peak height to baseline ratio mctiiwi
was used. When positive results were obtained, the fluorescence bands at hU.
410, 419, 427, 433 and 455 m/t were quite distinct.

As a result of several runs, the original aqueous distillate (D), was found to be
free of BP , whilst the charred residue contained 0*2 ng./lOO g. corrcspondiim to
0-07 pg./lOO g, of starch.

That the method has validity Avas also shown b}’’ the addition of 5 //g. of
BP to the methylene cliloride extract of a pjwotysed starch residue (from 30n g.
starch), and approximately 3 /ig. of BP was recovered. Since the amount of
BP originally present is about 0-2 //g. (see above) this represents a 56 per rent
recoA'^er3^

The amount of BP found (0*2 /eg.) is essential!}^ the same irheii benzene replaces
methylene chloride as solvent, and altogether the total experiment of pjToly.siii2
starch and estimating the BP has been done four times with both “ clmr " and
distillate (D) vdth essentiallj’' the same result. Though tiie distillate (D) is
extremely fluorescent no BP has been detected in it, though a 60 per cent recovery
was made ndien 5 /eg. of BP v^as added to it. No BP was detected wlien the whole
procedure was carried out starting by extracting unheated starch with motliylcnc
chloride.

DISCUSSION

The ydeld of BP ivas very minute, being 0-07 /eg. 1 100 g. of starch, much lower
than that of Gilbert and Lindsey (1957) who obtained 17 /eg. of BP per 100 g'. o
starch which had been heated to 650° C. This ydeld is probably little higher than
that of Kuratsune (1956) ivho reported 1 to 7 /eg.flOO g. of “ char ’. He used a

gas heated asbestos surface. «f

The fluorescence method of detecting BP is so sensitive that there is c ar g
contamination from outside sources. Hence a blank e.xpenment wiffi ‘ ^
amount of starch and treatment AA-ith the same amount of so "nrp cnt

starch was heated, aa^s carried out. No detectable amount of Bl P ; , ;
though less than 0-01 /eg. of BP per ml. of BP solution can [j;,

process used. MoreoAmr it is significant that the distillate, aaIi . p
same extraction treatment as the residue, gave no ^

It is unlikely that atmosplieric contamination is in question ,, ]

carried out in 1 iabomto.,- iom. 20 ft. above the br»t,- f™™;;”

some 400 yards from a busy motor road in a relati e y j.,,,,

The minute amount of BP found, corresponding to 1 g. of J 1 Iron
tons of starch, may have no practical significance. Unmus
pAwolyfsiS products have so far failed to show . thoiK'h iiflerjUAtr

Srried oM In the Melboiu-ne Univereity Patholoe' 'jj i" , ,«,^,'allr

Sete ha° e not yet been made rrith the relatively large amoun.e of ..pP

CAECIXOGENS FOUMEP IN THE HEATINC OE EdOl) STri'ES '2W

aromatic tjiies rvliich are dilViciiIl. to soparalc from tho HI’. Xt'VcrllK’lcsH llio
production of traces of BP at- the ])rcvio\isJy nnrcconlcd low tcunjuTat urc <if
370-390° C. may foreshadow the production of larger amounts at the same
temperature in actual cooking operations where the starch is exposed to oxiilation
and also the effect of other foodstuffs mixc<l with it. .Such experiments an* being
made.

.Sl'MM.VUY

Commercial starch has been " destructively distilled ” at atmospheric pressure
and at a temperature of 370-3510° C. Air was e.^sentially excluded. 'I'he charred
residue was found to contain 0*2 //g. of benzopyrene ])er loo g. " cliar ". 'I'ln*
significance is nnlcnomi.

Tlianks are due to Professor A. N. Hambly for advice, and to tbe .Anti-dancer
Council of Victoria for a grant. The microanalyses were carried out- by Dr.
Zimmemann and his staff.

REFERENCES

Bailey, A. S. axd Case, J. R.— (10,58) Tetrahalrnn. 3. 1 13.

Bailey, E. J. axd Dl-xgal. X.— (1958) Brit. J. Cmicr.r, 12, ;14S.

^bes, SI., Hopp, K. AXD Si-LA. J._(10.5-t) Csl. Onl.-ol., I. 25-1.
tiESEE, L. F.— ( 1957 ) Separatum Festschrift Arthur Stoll.

S- and Lixdsey, a. .1.— (19.57) Brit. J. Cancer, II, 398.

Y, A. t/.— ( 1955 ) Gastroenterology. 28, .345.

m-AWAY, E. L. AXD Len-dsey, A. .J.— (1958) Brit. vied. Bull., 14, 129.
toBATsirxE,M.-(1956) J. not. Cancer Inst., 16. 1485.
mm AM) HmEPEE, W. C.— (1958) Ibid.. 20, 37.
wT’ Arch. Sci. phys. nat., 46, 5.

^ek, D. j. asd Muller, K.— (1957) Roezn. Technol. Chem. Zywnnsci,

2 . 21 .

302

BACHEL STEIBMVEBBLOWSKr

Abnonmlities Detected at Autopsies of 54 Rat.^
Exposed to T agmal Benzopyrene Painting During the Oestrus Plum

Lesion

Polj'pus in horn .

C 3 ’St in hom .

Manunarj' adenofibroma

All over average .

Jfumber of
paintings
77
37
53
77
40

Duration of
experiment
(montlis)
24
IG
26
24
17

DISCUSSION

As can be seen, cancer of the cervix developed in 2 out of 53 rats painted diirin"
tJie (^oestrus (4 per cent). This is of particular interest because, as mentioned
previously (GJucksmann and Cherry, 1958) rats appear to be rather resistant to
the chemical induction of cervical cancer bjr painting. Furthermore i rat
developed an angio-epithelioma of the vagina. Other abnonnalities of interest
include 1 case of leukaemia, 1 case of bile duct carcinoma and 6 cases of mamtnar}'
fibro-adenomata. By contrast no malignant neoplasms either of tlic cervix or
vagina or at distant sites were noted in animals painted during the oestrus plin.<e.
From the above data it would appear that painting during the dioestnis j)iin?e
results in a higher incidence of tumours, both malignant and benign than paiutini:
during the oestrus phase. These findings ma}'' be due to several factors.

(а) The basal and regenerating cells may be particular!}" sensitive to carcino-
gens as shown by Breedis (1955) who found that the undifferentiated epithelium
of regenerating rabbit skin was higlily susceptible to chemical carcinogens. Or,
the keratinized epithelium may be resistant to such treatment as shomi hy
Twort and Twort (1936) who failed to produce tumours of the soles of foot of mice
that v"ere kept on plates smeared wdth carcinogenic oil.

(б) The duration of exposure of the basal cells, rather than their susceptilu’lit}
might be a factor in carcinogenesis. This aspect has been investigated b\
Berenblum, Haran-GIiera and Trainin (1958) with reference to the “hair cyclf'
effect. ” These authors found that the increased incidence of skin tumoiirj- m
the mouse when paintings rvere carried out during the resting phase of tlie liJur
e}"ele tvas not due to a hypersensitiveness during that particular phase but to a
difference in retention of a sufficient concentration of carcinogen. Similar
carcinogen may persist in the basal cells of the cervix for a mucii longer perux
than in the superficial, keratinized-cells receiving identical treatment.

The “hair cycle effect” naight still be enhanced in the case of the gcmui
epithelium. According to Glucksmann (1945) mouse epidermal cells dillercnti,
and are cast off in approximately 21 days whereas

vaginal epithelium of the mouse takes about 5 days (Snell, 1941). - I.

EXPLAJVATXOX OF PL.-ITE.

Fig. I. — Cervical epithelium rat. Oestrus, x 120.

Pjc 9 — Cen'ieal epithelium rat. Diocstrus, xt20.

Fig.’ 3.-Early cancer cf the cervix, X 120 (14 paintings vith/n S meaths).

Stein-IVerblowslty.

IKDUCTIOX of CAXCEK of the LHHIVIX

Si ceS Um .ell result in'e greater inlcsity a.,,1 .lurat.un of es,.osun.

““ZSast paiutiug duriug tl.c oestrus phase alTecIs a relatively smaller
number of cornified ceUs The period of exposure is also greatly reilueed as a
number of these cells are cast off within a verj' short t unc

The distant lesions observed in the dioestrus group might, be explained b.-s tin
absorption of the carcinogen by the subjacent blood-vessels of the denuis- a
nrocess which is greatly facilitated when the epithelium is of low, dioestnis t ype.
During oestrus absorption may be greatly delayed, as the thielv cornilied epit helium
may form a barrier against the penetration of the carcinogen and as the caremogeu
incorporated in the superficial cells is eliminated when these cells are shed.

It maybe mentioned in this connection that similar results have been obtained
by treating the basal and keratinized cells on other sites. Thus carciimgens
applied to the injured gastric mucosa of rats induced 10 cancers among V.V.\
animals rvhereas application on the intact gastric mucosa did not. yield any local
gastric tumours among 66 controls, though .several distant lesions Imve been
obsen^ed (Stein-Werblowsky, unpublished data). This is also in accordance with
the findings of Huggins (1958) who induced breast tumours in rats by gastric
instillation of methylcholanthrene. No mention is made of a concomilaiit.
induction of gastric tumours. Similarly the application of dimcthylbcnzaiit hra-
cene on the shaved and injured intrascapular skin in mice yielded 69 warts, 8
carcinomas and 19 leukaemias among 99 animals whereas no tumours were obtained
by painting the soles of 14 mice with the same carcinogen (Stcin-\\’'crblowsky,
unpublished data ; Twort and Twort, 1936).

Kennaway (1955) has compared the induction of human cervical cancer to the
production of cancer in animals— w'hat he called the “ mouse jiainting theory ”,
the carcinogens being possible human carcinogens including smegma (Plant mul
Kohn-Speyer, 1947 ; Pratt-Thomase(aL, 1956 ; Heins, Dennis and Pratt-Thomas,
1958). Another potential human carcinogen, to our knowledge not yet investi-
gated, may be the human ejaculate. The prostatic and testicular secretions
contained therein may have carcinogenic properties analogous to the carcinogenic
effects of ovarian extracts. Testosterone for example has been found to induce
tumours in Laboratory animals (Horning, 1958). Exposure of basal cells to
such exogenous carcinogens is possible during the immediate post-menstrual period

SirtpSe animals Ko

tion goes t„go,l,er ,ith JnAistenee tt

r;,;on, Tout o“ U.o's? SS S <“'“)■ *■> othSTanS,

—I and Oaapev rL'LZo. £

304

RACHEL STEIN-AVERBLOWSKY

type among Jewesses but do not attribute it to the Jaws of religious abstention
as the majority are no longer aware of their existence,

I opinions new and more detailed inquiries aupear to

be called for. Information should be sought regarding periods of abstention
wnthm the monthly cjmle, irrespective of ethnic and/or religious factors, .A
relation between such anamnestic data and the experimental fincliiws of ibis
paper might then be established. °

strarMAKV

Benzopyrene was painted on the cervix uteri of a group of rats in the tlioestnis
and oestrus phases respective^. In the dioestrus group 5 malignant and 14
benign lesions rv'ere obtained in 53 animals whereas in the oestrus group of at
animals onlj'' 4 benign lesions developed. The possible relevance of these
experimental findings to the aetiology of human cervical cancer is discussed.

The autlior is indebted to Hr. S. S. Epstein for his help and advice in the
preparation of the manuscript. Gratefid thanks are also due to Dr. G. P. Chern-
for several histological diagnoses, to ]\Irs. P. David for skilful tecimical assistance
and to Mr. E. Sai^ill for the pliotomicrographs.

REFERENCES

Berenblum, I., Haean-Ghera, N, akd Trainix, N.~(1958) Brit. J. Cancer, 12. 4fd.
Bbeedis, C. — (1955) Proc. Amer. Ass. Cancer JRes., 2, 7,

Casper, J.— (1955) Schtoeiz. Z. Path., 18, 769.— (1958) Abstract, llh i»t. Canctr Cr»iy..

Land. p. 281. .

D0Jo^^CH, A. AND Grulices, S. — (1956) Excerpia med., Ams(. (Cancer). \ ol. 4. Al)'‘ir.u
699.

Gagnon, F. — (1955) Sc//?i’eiz. Z. Pafk, 18, 755. , r ,

Gardner, W. 0., Auden, E., SmTH, G. M. and Strong, L. C. (1938) J. Jmer. m '■
Ass., 110, 1182.

Gault, E. W.— (1955) Sc/meiz. Z. Path., 18, 732.

Gducksjiann, a. — (1945) Cancer Res., 5, 385.

Idem AND Cherry, C. P. — (1958) Brit.J. Cancer, 12, 32.

Gudwaraes, U. P. and Bbaz, T.— (1958) Abstract, lilt ini. Cancer Congr., bona, j . - ■
Hausdoeff, H. — (1955) Zbl. Gynak., 77, 1854.

VON Haaai, E. and Scarpelli, D. G.— (1955) Cancer Res 15, 44.)

Heins, H. G. Jr., Dennis, E. J. and Pbatt-Thojias, H. R.— (19o8) Amc . . ■

Qiniec., 76, 726. ^ r, T /'r,„rrr 9 .'I')'’.

Hochmann, a., Ratzkowski, E. and Scheeiber, H. (195a) ri . . .

Horning, E. S.— (1958) Ibid., 12, 414. r j i

Huggins, G.— (1958) Abstract, 7th int. Cancer Congr., Land. p. i.

Kennaway, E. L. — (1955) Brit. med. J., h HO). w — ( 1058 ) Giaw f’'' -

Koprowska, I., Bogacz, j., Pentikas, C. and Stypulkor ski, \\ • ( • •

Murphy,’ E. D.— (1953) Amer. J. Path., 29, GOS.— (1958) Abstract, ith mt.

Conor., Load. p. 191. , „ „ ,o 1

Ober, W. B. and Reiner, L.— (1955) Schm^z. Z. Palh.,_i8, //4.

Perry, I. H.— (1936) Proc. Soc. exp. 35, 3-o.

Plaut a. and Kohn-Speyer, A.— (1947) Science, 105, 391.

IKDUCTIOX OP CAPJCKU OF TllK Cl'.UYIX

:ior.

Pratt-Thojus, H. R., Heins, H. C., Latham, K.. Dennis. K. J. and MrlvcH. 1'.

(1956) Cancer, 9, 671.

Reagan, J. W., Wentz, W. B. and j\LvcmcAo. N.— (I!).');')) Arch. Path.. GO. •1.51.

Runge, H. and Zettz, H. — (1958) Abstract. 7//i ini. Cancer Coiujr.. h>wt. p. ‘290.
ScARPELLi, D. G. AND VON H^mi, E. — (1957) Amcr. Path., 33. 10.59.

Snell, G. D. — (1941) ‘Biology of the Laboratory Mouse’. I’liilndelubiii (Hlahi'.lon).
p. 78.

SoESBY, M. — (1931) ‘ Cancer and Race '. London (Bale & Son).

Symeontdis, a. — (1951) Acta Un. int. Cancr., 7, 125.

Towne, J. E. — (1955) Amer. J. Obsicl. Gijnec.. 69, 607,

T\\’obt, j. M. and TSvort, G. G.— (1936) ./, Palh. Bact., 42. 303.

Vellios, F. and Geifein, j.— (1957) Cancer Jfc.-t., 17. 365.

Wyndee, E. L., Cornfield, J.. Schroff, P. D. and Doriswame. K. R —(19.51)

J. Obstet. Gynec., 68, 1016.

THE EFEBCT OF IHCUBATIOK OH THEEE

ttoioue viruses

H. A. DRAYTON

From the British Empire Cancer Campaign Unit, Agricultural Research Gonmil
Poultry Research Centre, West Mains Road, Edinburgh, 9

Received for publication April 22, 1960

1 ni tliermolabile character of chicken sarcoma I filtrates has been noted since

or-on^c’ Oliver, 1919). Later, it was found that incubation

at S i C. for 24 hours resulted in a rapid loss of infectivity even in filtrates of
Jugh titre and that inactivation was invariably complete after treatment at
55 C. for 15 minutes. Mueller (1928) first mentioned G 3 ’'e’s finding tiiat fiiis
inactivation of Rous I filtrates at moderate temperatures could he prevented for
periods up to three daj^s by the addition of HCN.

Gye (1925) ascribed tliis phenomenon of inactivation to the oxidation of a
“ specific factor ”, but later inclined to the view that inactivation was due to
“ proteolytic ferments in the filtrates acting on a non-living protein specific
factor ” (Gye and Purdy, 1930). In the light of Oppenheimer’s finding tliat
substances like HCN although powerful anti-oxidants, had little effect on proteases
(Oppenheimer, 1925), Mueller (1928) adhered to Cj’^e’s earlier theorj' that inactiva-
tion was a result of oxidation of a specific component of the virus.

Pirie and Holmes (1931) argued that since incubation at 37° C. even under
anaerobic conditions resulted in a loss of potencj’- of Rous I filtrates, atmosplicrie
oxj'^gen need play no part in any oxidation which is assumed to accompany Kous 1
virus inactivation. They suggested therefore that some oxidiser-catalj'st system
was closely attached to the virus itself, while admitting that it had not been
possible to separate it from the virus nor to identify it chemically.

With the exception of Rous I there is no information on the effect of incubafimi
on the avian tumour viruses. It was thought that a comparative study on tiic
effect of incubation at 37° C. for varying periods of time on the “infectivitv o
partially purified Rous I, jMHj (Murray and Begg, 1930), and PRCj (Carr am
Campbell, 1958) virus preparations might be of interest, if an attempt were nima
to correlate any effect on potency noth virus material liberated and detectc ’J
u.v. spectrophotometric analysis.

JIATEKIAIS AND METHODS

Preparation of normal cell particulate suspensions and of vi rus suspensions

Suspensions of cell particulates from normal fowl
from a non-filterable fowl sarcoma, GRCH 16 (Peacock

partially purified virus suspensions from Rous I, i mf^dificrl

prepared by the method originally de\used by Carr and tissue

by Bather (1953). In each series of experiments the same ^ suspension--,
was used as starting material for the preparation of each o

ISCUBATION

or mvi. TrM<n-n

vmrsr.s

UhT

Biodssuy . Urfwvn iin**

Chickens from the WgWy siisccp n e in of tin' tlin-i' vitm-

at the Poultrv Eesearch Centro wore nso,l for tin Ino.i. ..j

A-ims and aUow infectivity titrations to ho miul(> in }o«i'Jt ' .

Tnd no!nt (Carr and Harris, \051). The method of rnrlcor and Kivor.. ( 1 Ah. o a
used^to estimate the second figure in (ho end-, mint d.liit mn. ( .ronj.-. of 5 . in< - -

Avere used for each serial ten-fold dilution of virus ,, H,,.,

The initial untreated virus preparation Avns made m each o.im. hy n-u^p- n- in.
the “pellet” obtained after the final high-spooil centrifugation in as many ml. ...
Mcllvaine’s phosphate-citric buffer (pH 1 -2) us there were grams of ' ' wet f umour .
so that the concentration of virus before titration was snob that I ml. ^ lo'/.

tumour. “Infectivity” was always recorded ns m.i.d. g. of tumour (( >rr ami
Harris, 1951).

EXI'EniMKNTS

In eacb experimental series the “ infectivity ” of the three j.artiidiy puriii'*<l
A'irus suspensions was estimated by the bioassay method outliiu'd above, 'lb.-
non-infectu'e character of the GRCH and “ nonmd " eell jiarlie.iilate suspension"
Avas confirmed by inoculation of the undiluted preparations into t ehieks.

Equal volumes of each of the 5 suspensions snitahly dihiteil (Hi *) were sub-
jected to the folloA\-ing treatments ;

(1) Heated to 100° for 15 minutes.

(2) Incubated at 37° C. for 4 hours,

(3) „ 37° C. „ 8 „

(H „ 37° C. „ 16 „

(5) 37° C. „ 24 „

>! 37° C. „ 24 „ Avitb addition of M /I KC’.X.

(7) Maintained at 0° C. for 24 hours.

In each case after treatment, the virus/cell particulate was sedimented bv lii-b
speed centrifugation— 15,000 g. for 55 minutes— and the supernatant pipett'ed (dT.
diluted appropriately and examined in the u.v. spectropbotoincter (Unicam S1‘
oOO) at Avave-len^hs between 2400 A and 3000 A. Optical density was plolH-d
against a\ ua ^-length and the cun^es compared. The residue from ea'ch t rc-it meiit

Aseptic technique Avas obsen^ed throughout • tests fnr tBo r

^'Th^SJltf 7^ nutrient agar slopes incubated at 37° C., were negaUve

".enirotef .r J ctSTt ‘“'“f "« T ' ”

most detailed series are reported. ’ repetition only those for the

Injedivity

During the first four hours of incubation at a 7 ° r< ii,
dwrease in infectivity of each of the thrpp fp i s ‘ nn apprecia

i-c.. ts vSs :

308

H. A. DRAYTON

of 24 hours the wus preparations are completely non-infectivo. hoili,,. for r,

t loss of activity of tlie tliree virus proparatioiv'

Jlost of the infectivity of the three virus preparations is retained for 24 Imiirs

addition of KCN or after storage at (f V
(Tables I, II and III). Table lY summarises the 24 hour data alonolfroin otlirr
experiments.

Table I. — Effect of Experimental Treatments on Infectivity of Partially Pmftf,i

Eozts I Virus Preparations

Dilution of sample

and number of tumours

from 4 inoculations

“ hifocthhy "

of each dilution

of.iiiUnjit'

-A

(in. i.d. /n.

Treatment

10-'

10-2

10-2 10-'

luiumirj

Initially untreated .

4/4

1/4 0/4

0/4

111’-’

37° C. for 4 hours

4/4

1/4

0/4 —

—

ID'-’

37° C. for 8 hours

1/4

0/4

0/4 -

—

111'-'

37° C. for 16 hours

1/4

0/4

0/4 —

—

111’-’

37° C. for 24 hours

0/4

0/4 -

—

37° C, for 24 hours (+ KCN) .

4/4

3/4

0/4 —

—

in'-'

100° C. for 15 minutes

0/4

0/4 -

0° C. for 24 hours

4/4

3/4

0/4 -

, —

in’-'

.LE II. — Effect of Experimental Treatments

on Infectivity of

Parliattii Ihtriff'

MHn Vmts Preparation

Treatment

Initinllj^ tintreated .

37° C. for 4 hours
37° C. for 8 hours
37° C. for 16 hours
37° C. for 24 hours
37° C. for 24 hours (+ KCN)
100° G. for 15 minutes .

0° C. for 24 hours

Dilution of sample
and number of tumours
from 4 inoculnfions
of encli dilution

m-'

10-2

10'*

10-2

4/4

1/4

4/4

3/4

0/4

4/4

2/4

0/4

4/4

1/4

0/4

—

•

—

4/4

0/4

—

4/4

1/4

" Infi'clivity "
of WtUI'l"
(m-i.il./K-
ttunmirj

10 '-'

111 ’'

111 ’’*

111 '-'

lu'-'

10 '-’

Table III. — Effect of Experimental Treatments on Infectivity of Partially l^'':'

PRC^ Virus Preparation

Treatment

Initially untreated .

37° C.'for 4 hours
37° C. for 8 hours
37° C. for 10 hours
37° C. for 24 hours ^

37° C. for 24 hours ( + KCK)
100° C. for 15 minutes .

0° C. for 24 hours

Dilution of sample
and numlier of tumours
from 4 inoculation.s
of each dilution

j — — ^

10 -'

10-2

10 -'

10 '

4/4

3/4

0/4

4/4

2/4

0/4

(tj-i

4/4

0/4

0 /*

1/4

0/4

—

4/4

1 /'

0/4

4/4

. 1/4

0/1

•- Iuf--<-tivi','
(if l-.lOlf''-'
(in. i.d. /-

111 "

p,'.'

JO"

p.’’

(■

JO"

IKCUBATIOX OF FOWL Ti;Mori{ VIIU'SKS

:(0!i

Table Iv. — Effect on ‘‘ hifcctiviln ” of Tumour Virus Suspnislnus aiul Coutrots
Incubated at 37° C. for 24 hours. Compared u-ith Optical Ihusitij at 2S(l(l . f of
Material Liberated in Four Scries of Experiments

Experi-

ment

Dous 1

Decrease in infect ivity
(log units)

. 2-7

Optical density
at 2S00 A

. 0-10

. Decrease in infect ivity
(log units)

. a-S

Optical density
at 2S00 A

. o-3r,

. Decrease in infectivity
(log units)

. 4-3

Optical density
at 2S00 A

. 0-28

. Decrease in infectivity
(log units)

. 3-0

Optical density
at 2S00 A ■

. 0-15

Xnniiiil

r-II

AIH;

riu',

Hons 1
(-•• KfN)

Jiartieii.

Iat*'s

CItCM

4-7

. 4-3

. 0-4

— .

0-2f>

. 0-20

. 0-02 .

0- 1

o-os

3-7

. 2-.-.

. O'fi

—

OIS

. 0-13

. 0-01 .

0 •((.■) .

0-03

3 -,

, 4 • ft

. 0-ti

—

013

. 0-22

. 0-02 .

0 -os .

0-0.7

'1 • .7

. 3-7

. 0-3

—

0-34

. 0-2

. 0-01 .

0-1

<t-0.7

results, and lurrival 1 6 hours can bo cstnnatccl from tlic biossav

compare the rates of death of rS W curves it is i)o.ssibIo to

of these experiments In under the conditions

approximately 324 minutes (comnarerl'^ ^ for Rous I virus of

experiments (Rubin, 1955)) for of 19 i' I-i'Iun in his

1, 1900)) tor of 12 hours and 6-3 Iiours for PRC, (Rig. ;{).

Ultra-violet absorption
The ' 'fi

tliat repreSitcd L Rig°i 2^ spectrum examined from 2400-.3000 A is

htee virus preparatioS'inonbrted « 37?rrielS ^ f-1

... region 2750-2800 A and is vpt-, matenal -which absorbs strongly

incubated at 37° C suspensions of normal cell narHp

S ab.o,^«o„

M|(||t:Al hHl'tlly

IKCUBATIOX (ir FOWL Tl-MOFU VIIU’SF.S

partially purified Rous I, MH and PRC ^ ^ ■" i'>rco(ivi(.v of

a period of 4-24 hours is inciihalpfl af, Ilf' C

of protein-like material ‘in thnT^ ? ^ orincroasinK anionuN

manner do not yield material wirn propnrntiona treiilcd in the sain*.

G for MH, aM PRC wind) ensues after

“"'EOMion bt7v2 7 oxidaeion avh,“h l,.u

m Viewer rile 1,^ o«W m ^ay affect its intemalf.x tA ’molecular

-3fed in the spectra of the mate^nUn “I’ Jio'vcver,

>e deeonu.n=a,-„ suggested hv n.r^ ,n... proteases

'"°’°g'cal matLiak fAf P^^ein is kno^L^ £ ^ 1930).

‘i’^'^^ated in^tw'’ q«itr possible ff !?

2, ^om jmtoWto

812

H. A. DRAYTOIC

experimental procedure (e.g. total volume of fluid) and ti-.^
rat^ of decomposition may then vary from those reported.

The absence of detectable nucleic acid in the supernatants after niiv of tb'
treatments would suggest that the protein-like material liberated diirini: ifiaetiva-
tion, IS not that which is bound to ribonucleic acid in the nucleoids of Hons 1
virus particles (Epstein, 1958), but is derived from some part of the e.xtenial nut.

suaraiARY

1 . Experiments are described in ivliieh the effect of incubation on the biolryii'.sl
activity of Rous I, MH, and PBC 4 purified virus prejiarations, as determined bv
bioassay methods, is studied in relation to virus constituents liberated, as de-
termined by u.v. spectropliotometi^r.

2 . Infectivitj'' of Rous I, MHo and PBC 4 virus preparations decreased to nro
after incubation at 37° G. for 24 hours and after boiling for 15 minutes,

3. The decrease in infectivity of the three virus preparations incubated at
37° C. for various periods up to 24 hours was paralleled by the lil)eration of
increasing amounts of material, which absorbs strong)}' in the ti.a’. at 27r)(t~'Jsoa A
and is very likely protein. Since no such material was detected in GHCH Id and
normal cell particulate controls, it is concluded that the protcin-like nuiterial «;u
derived from the virus particles.

4. The loss of infectivity which ensues on incubation of Rous I, MKj and rih'i
virus preparations is associated not merely with simple oxidation l)iit hyactm}
decomposition of constituent xdnis protein.

All expenses in connection with this work were borne by the British Km)iiri'
Cancer Campaign.

REFERENCES

Adams, M. H.-— (1948) J. gen. Physiol, 31, 417.

Bather, R.— (1953) Brit. J. Cancer, 7, 492.

Cabr, j. G. and Campbell, J. G.~(1958) Ibid., 12, 031.

Idem AND Harris, R. J. C. — (1951) Ibid., 5, S3.

Epstein, M, A. — (1958) Nature Bond., 181, 1808.

Gye, W. E.— (1925) Lancet, ii, 109. „ ,

Idem AND PuBDY, W. J. — (1930) Brit. J. exp. Path.. 11, -8_.

Mueller, J. H. — (1928) J. Med., 48, 343. , r,,, /•,/ si

Murray, J. A. and Begg, A. M.-(I930) Sex. Rep. Imp.

Oppenheimer. C.— (1925) ‘ Die Fermente und ihre wirKimgcn . bmj.M. ((nor^

5 th ed., Vol. I, p. 75. ,, , no

Parker, R. F. and Rh-ebs, T. M.— (1930) J.exp. Med., 64 43. ,

Peacock, P. R. and Peacock, A.— {I9o3) Rrd. J. '

PiRiE, A. AND Holmes, B. E. — (1931) Brit. J. exp. Path., 12. - .

Rous, P., Robertson, H. AND Olix’eb, J. — (Htlit) d. ery. ^ a ■,

Rubin, H.— (1955) Virology, 1, 445.

abnomial distkibution of nucleic acid in

TISSUE CULTUBB CELLS INFECTED WITH BOLYOMA VIRUS

A. C. ALLISON AND J. A. ARMSTRONG
From the National In.stUntc Jor Medical Jia^carch, Mill Hill, l/nuhm. .\ .H .<

Received for publicfttion April 13. IPfiO

The polyoma virus, originally isolated from mice, has been sliown lo profhu-c
multiple tumours when inoculated into new-born mice and hainster.*i (otouai
Eddy and Borgese, 1958). Growth of the virus in mouse cmliryo t issue eultnro.s
is accompanied by a regular cytopathic effeett notably clumping of and
detachment from glass surfaces (Eddy, Stewart and Berkeley, lOoS). J lie
fluorescent antibody technique has shown that in polyoma-infcclcd (i.ssue
cultures of mouse embrj’o cells viral antigenic material develops first, in the host
cell nuclei and appears later in the cjdioplasm (Henie, Ueinhart and Rodriguez,
1959). Electron micrographs of ultrathin sections of tissue culture cells infected
udth the prototype SE and Mill Hill strains of polyoma virus have shown large
numbers of virus-like particles, mosttyin the nuclei (Bernhard, Febvre and Cramer,
1959 ; Dmochowski, Grey and Magee, 1959 : Negroni, Donrmashkin and
Chesterman, 1959).

Evidence concerning the nucleic acid component of polyoma virus has been
conflicting. Cytochemical changes in infected mouse Ijnnphoma cells were
reported by Love and Rabson (1959) who used the toluidine blue-molybdate
and Eeulgen techniques. Cellular enlargement was associated with the appearance
of abnormal nuclear vacuoles which developed a staining reaction for ribonuclco-
protein. From these findings, it was inferred that normal nuclear mechanisms
of ribonucleoprotein synthesis may be diverted into the proce.ss of virus replica-
tion. No evidence of an increased synthesis of deoxyribonucleic acid (DNA)
was foimd. On the other hand, Di Mayorca et al. (1959) have very recentlj'
reported that virus-free nucleic acid extracts of polyoma-infected tissue cultures
could reproduce the typical infection in other tissue cultures. The infectivity of
the extracts, unlike that of intact virus, was abolished by incubation with
deoxyribonuclease. Ribonuclease had no effect, and it was concluded that poly-
oma is probably a DNA virus.

In ^new of this discrepancy it may be helpful to record briefly some observations
recently made in this laboratory which are at variance wdth the previously
published cytochemical data. The technique employed has been the acridine
orange polychromatic fluorescence technique for differentiation of nucleic acids

Cells

JLATEKIALS AND METHODS

314

A. C. ALLISOK AICD J. A. ARMSTROXf!

Gey s sahne 80 parts ; Jacto}bHmin hydrolysate (5 per coat of stock! 10 ■

calf serum 10 parts; penicillin 100 units per ml. : streptomycin 100 a- iJr ml ■

feed m 2 ml volumes containing 10« cells into tcst-tubc.s with covenditK \ft,.r
3 days growth m the same nutrient medium, the medium was rciiLci aitli
another contaming 5 per cent calf serum and the vini.s inoculum. A str.sia ,4
L cells (obtamed from Dr. J. C. X. Westwood. Microhiologiea! Itew.m.f,
itstabhshment, Porton, Wilts.) was maintained in a medium coii.si.stim' of (lev's
salme 70 parts ; calf serum, 20 parts ; yeast extract ( 1 jicr cent of .slock)" 10 jurN '■
l^talbumm hydrotysate (5 per cent of stock) 10 parts ; and antihiotio- as ahovo,’
colls "woro t-riinsfoircd to test tubes coufiiinin^ covcr’Iij'*
grown and infected in a medium containing 10 parts of calf .scrum.

Vims

SE polyoma virus, batcli 3E29~4, obtained from Dr. K. E. Eddy, Xafion,)!
Cancer Institute, Bethesda, Md., U.S.A., was propagated in mouse cmhrvo cc!)
cultures in flat bottles. After 7 days’ incubation witli two clianges t)f nirdinm
the cells were removed and treated with fluorocarbon (Giardi, tOriO) to jircpiri-
stock virus. Virus was titrated by haemagglutination of gninca-pig rclb and
infeeti\ntj’' in mouse enbrjn tissue cultures (Rmvo ct a/., 105!)).

MicroscoTpy

Coverslips from control and infected cultures were removed at daily intervaU
up to 8 days after virus inoculation, rinsed in tris-buffered Goy's .solution |pli "•!)
and fixed in absolute alcohol. The method of staining cover.dip eiiltnn-s with
0-05 per cent acridine orange (C.I. 788) inacetatc-HCl bulTcr (pH 2-7) wusde.'Crih''*!
by Armstrong and Hopper (1959). Examination was by dark-lieid (hiore.stvnrr
microscopy, using the bine-violet emission of a high pressure merenry \M))’>nr
lamp for activation of fluorescence. Other cultures were fixeii for -’i) mimit*'-'
in 1 per cent osmium tetroxide buffered to pH 7-4 (Palade. I9i>2) ; ufter rio-ioe in
Gey’s solution they were allowed to .stand overnight in 70 per cent etlmiif)! a.'id
finally mounted in water for examination by pha.se-conlrasl mier.i-eojiy.

Ihi: !
lift!*

'I.f!

OKSEKVATIONS

Vims yield , ,,11,

The appearance of infective virus in mou.se kidney ceils and ciiltur.'

after inoculation witli 10^ TCD,o of polyoma viru.s is .slmwn m !• ig. 1 . \ cry

virus could be recovered at 24 hours. There wa.s a con.^derablc
associated vims between the second and third days after ,

little virus liberation into the culture medium, from Ibe bi d t t/ .-
day after infection there was a slou^r increase

increase in the virus liberated into the medium. ‘ ,.f - ;

more than one-tenth of the total virus m the ])reparation. n n ln r ^

“eUs virus jdeld was low at all time.s and only a small proportion of .< I!

morjihological changes.

or .1.0 .o„.o of),

under the low power of the micro-seopc shoue<! n 1 fh, ,

, ,f

OTCbiiiv: AV.V1J .f v,^U\''

s -“1 =S53^|S i-iSS

fifth and eighth days after infection.

i'lG.

1 -Appearance of cell-aseociated virus (continuous line) nnd vin.s in tu.-lniin (int-rmiu-l
line) in mouse kidney cultures infected uilh [jolyomn vini'-.

Cytology

The fluorescence of cells in uninfected mouse kidney cell culture.^ after treat-
ment with acridine orange is reproduced in black and wliitc in Fig. 2. In aettial
specimens the nuclear margin and chromatin network (including ii»el(>i)liis-
associated chromatin) emitted the greenish-yellow fluorescence wliicli is typical
of DNA-containing elements. This contrasted sharply with tlie orango-rwl coknir
of the nucleoli themselves, and of the ribonucleoprotein whicli is scatt ered t hrougli ■
out the cytoplasm.

No distinctive cytological changes were observed in these cultures nji to k
daj's after inoculation uith polyoma virus, but on the fourth day there were
multiple foci in which cells had become rounded and were easily detnclied from
the glass surface, From this time up to the eighth day of infection an unusual
t3rpe of intranuclear structure avas detectable in many of the cells close to where
rounding up and cell detachment were in progress. It was found only occasionallv
in cells of the intervening, and intact, parts of the cell sheet. The material coii-
cerned gave an intense greenish-yellow fluorescence of the DNA tj^pe. ]n -t
day-infected cultures it was mostly located in one or more discrete centres within
tlie nucleoplasm ; but by the eighth day a high percentage of the affected nnclei
were almost completely filled. Detection of the earliest stage was made difficult
by the presence of rather large chromatin granules in the normal cell nuclei tliis
lemg a constant feature of many uninfected murine cells in ti.ssue culture (Fie 2) •
some nuclei shomng minimal alteration of the DNA pattern after infection may

316

A. C. ALLISON AND J. A. ADMSTEOA'G

liave been overlooked on this account. Nuclear frac^mentation wac rar„i,

AS Illustrated jn Pig. 3-6, tlie appearances were verj^ different from those of -
non-specific degeneration or of pyknosis. The figures shoir four tj^icaUv affected
nuclei arrangecl in order of increasmg abnormality ; the black and white prints
ere prepared from original colour transparencies. The positions of nucleoli
Identifiable because of the red fluorescence of their RNA, are indicated as a miide
in hig. 2 and 4 ; the remaining nuclear fluorescence n-as entirely oftheDRA tnie
In addition to nuclear changes there was vacuolation of the c 3 doplasm in some of
these cells (Pig. 3 and 6). No regular change was noted in the structure of the
nucleoli or in their fluorescent intensitJ^ None of the infected mouse kidney cell
cultures showed evidence of NNA-containing material in the cj-toplasm.

Almost identical changes were found in the potyoma-infected mouse embryo
cultures ; and a few scattered cells with the same abnormal patterns of intra-
nuclear DNA were present, in the corresponding cultures of “ L ” strain cells.
In the latter, Jiowever, occasional cells with abnormal nuclei had in their ci1o-
plasni a prominent paranuclear mass of greenish-yeUow fluorescent material
(Fig. 7). This was quite distinct from the dull green fluorescence sometimes found
in a hjqoertrophied Golgi zone of cultured epithefial cells. It was also the only
indication of a cjdoplasmic DNA reaction in tlie potyoma-infected cultures at
our disposal.

Phase-contrast studj’- of the osmium-ftxed kidney’’ cell and mouse embiyo
cultures confirmed tliat nuclear changes of a peculiar l^d occurred in the vicinity
of the focal cytopathic effect. It has been shown that osmium fixation avoids
structural artefacts which are sometimes brought about by’’ the moie commonly
used histological fixatives (Strangeways and Canti, 1927). Moreover, cytologieal
details may’’ be revealed by^ phase-contrast with greater clarity than is usually
possible in the equivalent hving cell. A normal cell nucleus is shovTi in Fig. S ;
the nucleoli are very^ dense relative to the surrounding nucleoplasm, which is
faintly^ stippled. The tyqie of nucleus seen in Fig. 9, however, occurred only after
the fourth day’^ of infection irith polymma virus. Irregular patches of soraeirbat
lower density^ than the nucleoli, and of varyring size, are present in the
plasm ; their appearance resembles closety that of the material revealec i
fluorescence microscopy^ after alcohol fixation (Fig. 3 and 4). In \dev o us
it seems likely that the distribution of nuclear contents shown by the fluorescence
microscope was not, in this case, much influenced by'^ fixation artefact.

EXPLANATION OF PLATES

Fio. 2-7 are fluorescence photomicrographs of cells in tissue bv

orange technique. Prints were made from colour transparencies b

arrows in Fig. 1 and 3) were red (RNA), the remammg nuclear fluorescence greeni. .

(DNA).

Fig. 2. — Uninfected mouse kidney cells, x 120(X l-iSnev cells, between 4

Fig. 3-6. Abnormal patterns of nuclear DAA fluorescence m mouse

and 8 days after infection with polyoma virus, Xl200. fluorescence C daw after

Fig. 7.—“ L ” strain cell showing a ejdoplasmic mass of DAA-Ope fluorescence

infection with polyoma virus, X 1700. „ „r„ normal mouse kidney cell

Pig. 8. Phase-contrast photomicrograph, showmg the nucleus of a n

in tissue culture. OsOi fixation, xlSOO. ■r.fl.ntpd S davs previously

Fig. 9.— Similar preparation to Fig. 8, but from a culture infected 8 days p

polyoma vims. OsOi fixation, XlSOO.

British Journai. of Cancer.

Vol. XIV. Xo.

ong.

Allison and Annstn

nucleic acid IK POLYO^U ^^RUS IKEECTED CELLS

COMJIENT

The cytoohemical ch.ages reported here are rcminiacort "f

“srSd

chtng?”™tS‘Siated Sto rmaLSStortion of m^lonr i"orpli'>j»B;
Tvhich is so Uniical of adenovirus infections and vliich is grcatlj influenced
alcohol fixatira. The present findings would appear, therefore, to he consistent
^vdth the repheation of a DNA attus within the nuclei of susceptible murine clIIs.
Close inspection of the control specimens revealed no comparable DNA pattern, so
that the possibility of a spontaneous development of bizarre nuclear forms is very
unlikely. We interpret the present findings in polyoma-infectnd^ cultures ns
additional evidence for the view, expressed by Di Mayorca el al. (1950), that tlic
polyoma agent is in all probability a DNA adrus. _

The first indication of infection is the appearance of multiple foci of DNA-
containing material in the nucleoplasm. The findings suggest that these enlarge,
coalesce and eventually fill the nucleus. Cells at tliis stage of virus development
presumably correspond to those which have shovm large numbers of virus-like
inclusions when examined by electron microscopy. In infected “ L ” cell culture.s,
a few cells were seen -with DNA-containing material in the cytoplasm, suggc.sting a
discharge of virus nucleic acid from the nucleus into the cytoplasm, but in the
mouse kidney and mouse embrjm cells there were no signs of cytoplasmic DNA.
This might be related to the fact that moribund cells with large amounts of intra-
nuclear virus are detached from the coverslips before disintegration and release of
virus. Certainly the occurrence of recognizable cells in the medium coincided with
the appearance in it of detectable virus. In general, the fluorescence induced by
acridine orange staining is comparable with that obtained in the earlier stages of
infection by means of the fluorescent antibody technique (Henle et al., 1959),
but with the latter some cells in the late cultures showed cj’toplasmic fluorescence.
This might be due to the passage of virus from the nucleus into the cytoplasm,
but alternatively could indicate the presence in the cj^oplasm of antigen not
accompanied by nucleic acid and not therefore visualized b3’^ acridine orange.

A relatively long latent period and slow progression of infection with the poly-
oma virus appears to be the rule, and despite the use of relatively large virus
^ ® showed cytopathic effects and accumulation of

DNA contammg material m the nuclei. It is conceivable that other parts of tho
cell sheet were infected but transformed into tumour-like cells releasing ven^ littio
virus as suggested recently by Vogt and Dulbecco (1960). ^ ^

of Vitus n„d the appearance of cjdochemioal Sat

characteristic cj-topathlc effects. ^ minority of cells, showed

318

A. C. ALLISON AND J. A. ARMSTEONG

After infection the first change detectable in cells stained m'th acridine oranne
and examined by fluorescence microscopy atos the appearance of abnormal DM-
contaimng foci in the nuclei. At a later stage cells became detached from the
coyerslips and the nuclei of some AA'ere by then almost filled with the DNA-con-
taining material. It is concluded that polyoma virus contains DNA and multiplies
in the nuclei of susceptible cells.

We are indebted to ]\Ir. M. R, Young for taking the photomicrographs.

REFERENCES

Armstrong, J. A. — (1956) Exf. Cell. Ses., 11, 640.

Idem AND Hopper, P. K. — (1959) Ibid., 16, 584.

Ideyn AND Nin'en, J. S. F. — (1957) Nature, Land., 180, 1335.

Bernhard, W., Febatje, H. L. and CRAsrEB, R. — (1959) C.B. Acad. Sci., Paris, 249,
483.

Di JLiyorca, G. A., Eddy, B. E., Stewart, S. E., Hunter, W. S., Friend, G. and
Bendich, a. — (1959) Proc. nal. Acad. Sci., Wash., 45, 1804.

Dmochowski, L., Grey, C. E. and JIagee, L. A. — (1959) Proc. Soc. exp. Biol. A. I.,
102, 575.

Eddy, B. E., Stewart, S. E. and Berkeley, W.— (1958) Ibid., 98, 848.

Glvrdi, a. j. — (1959) Virology, 9, 488.

Henle, G., Deinhardt, F. and Rodriguez, J. — (1959) Ibid., 8, 388.

Love, R. and Rabson, A. S. — (1959) J. nat. Cancer Insl., 23, 875. _

Negroni, G., Dourmashkin, R. and Chesterman, F. C.— (1959) Bnt. med. J., ii,

Palade, G, E. — (1952) J. exp. J/ed,, 95, 285. mo

Rowe, W. P., Hartley, J. W., Estes, J. B. and Huebner, R. J.— (1959) Ibid., lua,

379

Stewart, S. E., Eddy, B. E. and Borgese, N. G.-(1958) /. nat. Cancer Inst., 20, 1223.
Strangeways, T. S. P. and Canti, R. G.~(1927) Quart. J. nmr. Sci., 71 1.

Vogt, M. and Dulbecco, R.— (1960) Proc. nat. Acad. Sci. 11 ash., 46, 366.

the eeeect oe hitroeurazone oh ^

^RX ORGANS OE NORl^IAL RATS AND RAJS

CARCINOMA 250

D. G. MOXTEMUBRO*

From the Chester Beatty Besearch IvstiMe hMe ofCnvccr Jiesearch :
B.oyal Cancer Hospitaly LondoHj o. .*1

Received for publication March 24, lOfiO

Peofotisd changes in the endocrinology of the host result from tlie growt h of
not only the so-called functional endocrine tumours but also from progressive
growth of non-functional tumours. Begg and Stewart (1052) and Begg (lO.m)
observed atrophy of the testes, seminal vesicle and prostate of rats bcanng the
AValker carcinoma. This atrophy could not be influenced by forcc-fccding
designed to maintain carcass weight of the tumour-bearer (Begg, 1058). They
found, however, that the atrophy could be prevented by the exogenous adminis-
tration of serum gonadotrophin and suggested that ; “ . . . the presence of a
tumour produces a deficiency of gonadotropliic hormone in the rat”. These
observations have been confirmed and extended by Haddow, Horning and
Carlton Smith (1957), who found that testicular changes were not as consistent ns
those reported previously and were restricted to an increase in number and lijiid
content of the Sertoli cells. Marked hypertrophy of the adrenals was a consistent
finding, and in some cases n^as associated with hypertrophic changes in the pitui-
tary gland. Atrophy of the accessory sex organs could be reversed by testosterone
propionate, serum gonadotrophin or by the subcutaneous implantation of pituitary
glands.

Nissim (1957) found that nitrofurazone, an anti-bacterial drug sometimes used
in infections of the urinary tract, when added to the diet of mice in concentrations
of 0-15-0-3 per cent resulted in atrophy of the spermatogenic epithelium of the
testes, interstitial cell hyperplasia, and hypertrophy of the seminal vesicle. The
effect was absent in hypophysectoraized mice, and he suggested that the andro-
genic action may be a manifestation of hyperactivity of the pituitary due to the
release from a pituitary inhibiting factor normally liberated from the germ cells
of the testes. Castration cells in the pituitary— an indication of hyperactivitv

reported by Nelson and Steinberger
o ?hat If f administration of furadroxyl, a drug having a similar action
to that of nitrofurazone. The present work deals with some efiects of nitro-
fiirazone m normal and tumour-bearing rats. Should nitrofurazone be shown
to have an androgenic action in the rat as it does in the mouse it would l

• British Empire Cancer Campaign Exchange Fellow, 1958-60.

320

D. G. MONTEMUREO

JIATEHIAIiS AND METHODS

Male albino rats from the colony at the Chester n i. t

kr* ? -

i.°“eS5“» Se ^ ‘° P““'

Table I. — Composition of the Diet

Constituent

Per cent
by weight

Wheat flour

68-6

Casein

H-5

lliik powder

8-0

JIargorine .

3-3

Bemax

2-5

Yeast

2-4

Cod liver oil

1-6

Calcium carbonate

1-3

Glaxo salt mixture

0-8

The dry diet was mixed with sufficient water to make a dough-like paste which was then fed to
the animals ad libitum.

The 'Walker carcinoma 256 serially transplanted at tliis Institute, grows verj'
rapidlj’- and m'thin 14 days may weigh in excess of 70 g. Accordingly, all animals
■were sacrificed by an overdose of ether anaesthesia 13 days after the subcutaneous
implantation of the tumour. At necropsy the testes, prostate, seminal vesicle
and tumour were treighed to the nearest 0-01 g. The seminal vesicle was stripped
of coagulating gland and ligated at its junction vdth the ductus deferens before
excision to prevent loss of seminal fluid. All tissues were fixed in Bonin’s solution,
embedded in paraffin, sectioned at 5 microns and stained wdth haemato.xylin
and eosin.

RESULTS

(a) The effect of nitrofurazone feeding on the testes and accessory sex organs of normal
male rats

Eighteen rats of the same age and approximate weight were used for tins
experiment. Six w'ere fed the normal basic diet and acted as controls ; the
remaining 12 were divided into two groups receiving the basic diet supplemente
with nitrofurazone 0-15 per cent in one, and 0-50 per cent in the other. ic
latter dose had to be reduced to 0-30 per cent after three days due to the death m
one rat and general physiological deterioration of the remaining five. A
days the experiment was terminated. The weights of the testes, semina ^ esic c
and prostate are shown in Table II. Nitrofurazone feeding resulted m an appreci-
able loss of bodj^ weight in both groups of experimental animals ; le ^ ■
weight loss was observed in those rats receiving the larger dose o
Although food consumptions were not recorded, daily observation o i < ^

gave the impression that the inhibition, of food intake was proper loni

ettect of EITROFURAZOXE

:v2l

loss, the ^veigMs of the organs was evident in all rats receiving

of the body weight at death. Testic of the drug. H istologieallv.

the drug hut was greater in those fed snermatogcnic epithelium (Fig.

rrSroct^S h?S^iV™However,

cells as described by Ishssim (1957) in the mouse nas not cMdcnt.

Table II.— TAe Effect of Niirofurazone Feeding onEoch/ Weight, Fc.dcs and
Accessory Sex Organs of Normal Male Jiats

Number

Normal diet . . . S

0-15% nitrofurazone in diet . 6

0-30% nitrofurazone in diet . 5

Death

Sctniiiftl

I’n'stnte

weight

(g-)

Testes

vcsitdo

(mp./ion g. body

weight)

522±21»

7264-28

209 4-20

105±0

366 ±10

377 4-27

34n±48

120± 10

«0.001)

(<0-001)

«0-02)

94±3K

307 ±13
(<0-001)

514 ±.62
«0-01)

224 ±30

* Mean ± standard error.

( ) Statistical probability of difference from normal diet.

The mean absolute weight of the seminal vesicles in the group of rats fed the
lower dose of nitrofurazone was greater than that of the rats on the basic diet.
When expressed as a percentage of the body weight this increase (140 mg.) was
statistically significant at the P <0-02 level. The increase in proportionate
weight of the organ in the group of rats fed the higher dose of nitrofurazone was
less obvious. Histologically, the seminal vesicles of the experimental animals did
not differ from those of the normal rats. The weights and histology of the pros-
tates of the experimental animals were not unlike those of the controls.

(b) The effect of nitrofurazone feeding on the testes and accessory sex organs of
bearing the Walker carcinoma 256

Thirty 11-week-old rats of approximately the same weight received subcu-
taneous implants of the Walker carcinoma 256. Eighteen were given the basic
diet, and 12 the basic diet supplemented with 0T5 per cent nitrofurazone ad
libitum. Twelve normal rats of the same age and weight fed the basic diet alone,
.served as controls Tliirteen days after the implant all animals were sacrificed
by an overdose of ether anaesthesia. The weights of the tumours testes and
accessory sex organs at necropsy are shown in Table III. Once again the mean
neights are expressed as a percentage of the final body weight. Grorvth of the

anto,, Smi?" “ok h Je Homing and

weight of the tumour. Fig 2 shows the^reT proportional to the

2 shows the relationship between seminal vesicle

D. G. MONTEMUBEO

:^22

Jo?' Nitrofurazone on the Testes and

Accessory Sex Organs of Tumour-bearing Male Rats

A^orinnl rats (basic diet) .
Tumour-bonrors (basic diet)

body weight
Number (g.)

. 12

Final Tumour weight

/n/ 1 1 ®

Tumotir-benrcrs (O'
furn/.ono in diet)

nitro- 12

492 ± 14 *
<01
413±19
<001
328J:22

(% body
weight)

13 - 8 ± 1-9

< 0-6

15 - 2 ± 0-8

Seminal

Testes vesicle Prostate
(mg,/100 g. body weight)

786±23
<0-3
844 ±3S
<0-001
461 ±10

2IS±13

<0-01

122^20

<0-7

110±14

J22J-9
<0''001
60 - 1-7

<fs

71 ±9

* Sfean ± standard error.

TUMOUR g.

Fig. 2. — Relationsliip between the weight of the seminal vesicle and the weight of the tumour
in 24 rats bearing the Walker carcinoma 256.

%veiglit and tumour u^eight in 24 untreated tumour-bearers (18 from this experi-
ment and 6 from the experiment described in Section c). Although the scatter
is great, a statistically sigm'ficant negative correlation (r = —0-664 ± O'll")
could be demonstrated between the two measurements. Roughly 43 per cent
of any decrease in seminal vesicle weight is associated with an increase in tumour
weight. .

The feeding of nitrofurazone to tumour-bearing rats resulted in body weig i
loss and profound testicular degeneration similar to that observed in norma
rats (Section a), but had no effect on the seminal vesicle and prostatic atrop 1
associated with the growing tumour. The drug had no notable effect on the ra e
of growth of the tumour.

Six tumour-bearing rats were treated daily with 10 mg. of
suspended in 0-4 ml. of arachis oil. Six untreated tumour-bearers and six n

effect of kitrofubazoxe

rats acted as controls, “nvs" Tlic'rcsnils of llii.s osi'ori-

n.e W„„tcr rosolto,,

Tannn W.-TJ. Mecl of m,rofurn.o„c

on tte Tedet ond Aecessonj Sa Organs of Tnmom lam in. I

Kormal rats . . • ^

Untreated tumour-bearers ^

Tumour-bearers (10 mg. nitro-
furazone daily for 10 days)

body rreigbt
Xumber (g-)

. 6 . 3Glil4»

P . <0-2

. G . 403 ±21

P . <0-0

. 406 ±0

Final Tumour woigbt

(% '>oib'

weight)

17-4±1-2

<0-5

14-2-^2-0

St'ininal
Testes vesiele 1
(mg.; 10(1 g. body w

I0S.7±41 17S±II

:n-oi

84 5 ±50
■tCO-0

880 ±.51

< 0-001

00-1-14

■co'-oi

124 ±8

roMtnt**

eight)

02-1 7
.''0-02
51 J II
-cd-5
o 't -1 4

* Mean ± standard error.

in marked seminal vesicle and prostatic atrophy. The epithelial limiig of the
seminal vesicle was altered from the high columnar type to a low cuhoitlal non-
secretory tj^pe (Fig. 3a). There was a decrease in seminal fluid and an increase in
the fihromuscular stroma of the gland. Atrophy of the testes was more iiot iccahic
than in the previous experiment ; however, sections of the testes stained with
haematoxylin and eosin revealed no obvious alteration in testicular structure.
Haddow, Homing and Carlton Smith (1957) have shorni that Sudan I\’ staining
of frozen sections of the testes reveals an increase in number and lipid content- of
the Sertoli cells in the tumour-bearer, which was taken as evidence of some
abnormality in testicular function.

Treatment with 10 mg. of nitrofurazone daily for 10 days had no effect- on
body weight nor on the weight of the tumour at necropsy when compared with
the untreated tumour-bearers. Hor were the testes of the treated group of rats
different from those of the untreated tumour-bearers in gross weight and in
histological appearance. In fact, the only notable effect of this treatment was the
almost complete restoration of the weight and histological appearance of the
seminal vesicle to that seen in normal male rats of the same age (Fig. 3&). Changes
in the weight aird histology of the prostate were less obvious. Thus the atrophic
changes in the seminal vesicle and to a lesser degree in the prostate, can be largelj'
prevented by the intraperitoneal administration of nitrofurazone.

Larger doses of nitrofurazone (30 mg. /day for four days) proved fatal to two
out of SIX tumour-bearers. The remaining four showed, at autopsy, profound
testicular atrophy, while the seminal vesicles were slightly but not significant! v
larger than those of the untreated tumour-bearers. The body weights and

SreaLrimp.

-UIOUUSSIUN

tho l«-dcpression of liver catalase aotivfty

..»ds. .trap,,, of «.e and

324

D. G. MONTEMURKO

the

cachexia, to mention but a few The nf f ^ .

previously-made observations of Beag (1955)

Haddow, Horning and Carlton

carcinoma 256 results m atrophic changes in the seminal vesicle and prostate and
to a less notable extent m the testes. Microscopically the accessory sex omans
hrSe heSit ’^f diminished or complete absence of secretion, reduction

I f f epithelium and an increase in libro!

muscular stroma. These changes can be prevented or reversed by the exot'enoiis

provided by the daily administration of nitrofurazone (Table lY and Fig. 36).

The androgenic action of nitrofurazone shown by Nissim (1957) to be present
m the mouse maj'' also be obtained in the rat. Both species respond to treatment
wth profound degenerative changes in the spermatogenic epithelium of the
seminiferous tubules, but hj'pertrophy of the aecessorj’' sex organs is less dramatic
than that described in the mouse. The reason for tin’s difference niaj’ be that
degeneration of the spermatogenic epitheljum in tie rat testes was not accompanied
^3' noticeable lij'perplasia of the interstitial tissue. Hyperplasia of Lej'dig-
cell tissue was more apparent than real, due to the large interstitial spaces left
by the shrunken and distorted atrophic tubules (Fig. lo). Further, mitotic
figures were not more numerous in the interstitial tissue of nitrofurazone-treated
rats than in that of untreated normal rats.

The results of these experiments allow some comment on the possible
mechanism of the androgenic action of nitrofurazone. Nissim (1957) suggested
tliat this action is attributable to increased pituitary gonadotrophin activity
consequent on the m’thdrawal of an “ inhibitory substance ” normally liberated
bjf the intact seminiferous tubules. In the experiments on the rat reported here,
the maximum hypertrophy of the seminal vesicle — or, more accurately^ the
greatest inhibition of seminal vesicle atrophy^ — was seen in those rats that did
not show degenerative changes in the spermatogenic epithelium (Table II')-
This would indicate that the postulated increase in pituitary gonadotrophin
activity following treatment vdth nitrofurazone is not necessarily dependent on
initial degeneration of the spermatogenic epithelium of the testes.

An adequate explanation for the failure of nitrofurazone feeding to prevent
atrophy of the accessory sex organs of the rats shown in Table III Js wanting
However, since administration of the drug by the intraperitoneal route did not
produce anorexia and weight loss, the severe under-nutrition associated witli nw
feeding experiments may have resulted in a pituitary gland refractory^ o any
stimulltory^ action of the drug. Mulinos and Pomerantz (1940 have compare
the hormonal imbalances of malnutrition to that of surgical hypophrsecton ,
and described this phenomenon as a dietary pseudo-hyTophysectomy. ^

Some degree of nutritional deficiency or metabolic alteration probab y p
major role on the atrophic changes seen in the accessory- oTa tumour,

lesser extent in the gonads associated -unth t In-

Hormonal imbalances may be effected by retention f (pm},

the pituitary- basophils in a similar manner to . to asseL tbc

and Pearse and Einaldini (1950) in semi-starved rats.

gonadotrophic content of the pituitary gland of tumour-bean g < P -

EFFECT OF XITUOFCRAZONI'-

:F2r.

and Cdochcmical ,ocl,„i,„ca T" '.r'uu.

seen in tumonr-bearing animals.

SUMMaVnV

1 The effect of iiitrofurazone on the testes, seminal vc.siele anti pro.st.ilo of
normal rats and rats bearing the Walker carcinoma 2ofi were .st iidicd.

2 blitrofurazone added to the diet of normal adult male rats in a concent rat mn
of o'l5 per cent resulted in body weight loss, profound degenem ion of the
spermatogenic epithelium of the testes and a slight, hypertrophy of the seminal
vesicle. Hyperplasia of interstitial testicular tissue was not. npiiarcnt •

3. Atrophic changes in the seminal vesicle consisting of a reduction in height
of the secretorj' epithelial cells, a reduction in the amount of seminal secretion,
and a notable increase in fibro-muscular stroma of the gland were associatcil with
growth of the Walker tumour. The degree of atrophy was signiticaiU ly correlated
with the weight of the tumour.

4. Addition of nitrofurazone to the diet of tumour-hearing rats failed to
prevent atrophy of the seminal vesicle and prostate glands. Degeneration of
the spermatogenic epithelium of the testes was similar to that seen in normal rats,

5. Intraperitoneal administration of 10 mg. of iiitrofurazone for 10 days
protected tumour-bearing rats against atrophy of the accessory sex organs.
This protection was associated with no abnormal change in body weight , nor in
weight or histological appearance of the testes.

6. These findings are compared with the previously reported effects of nitro-
furazone in the mouse. It is suggested that cj'tological studies of the pituitary
gland of tumour-hearing rats would provide a clearer understanding of the
hormonal imbalances associated with the growth of tumours.

The author wishes to record his admiration and respect for the late Ih-ofessor
E. S. Horning under whose direction this study w'as initiated. I am indebted to
Professor A. Haddow, Director of the Chester Beatty Research Institute, for his
kindness and consideration in providing facilities with wiiich to carry out this
work The excellent histological preparations are the work of j\Ir R J
McColloch, the photographs that of Mr. K. Moreman and the pliotocraiihic
department of the Chester Beatty Research Institute. ^

In f ® been supported by grants to the Chester Beatty Research

Institute (Institute of Cancer Research : Royal Cancer Hosnitall fmm rim vr v i

C»o„ I„st«e „t the NaWl I„,tit„Us of Health, U.S. pl“ He^^^

references

/uCin ANT^ fNT’P\VAT>rn A r\ _

5 , 1 .

hkm AXD Stewmrt. A G-_nQS‘)i r> n
Haddow, A., Honxmo E S 248.

87, 396. ’ ’ Caelton Sauth, N.

(1957) Schweiz, med.

Wsc

326

D. G. MONTEMUERO

Mulinos, M. G. and Pomerantz, L.— (1940) J, Nntr., 19, 493.

ISlELSON, W. 0. AND Steinberger, E.— (1952) Anal. Bee., 112, 367
XissiM, J, A.— (1957) Lancet, i, 304.

Pearse, a. G. E. and Rinaldini, L. M.— (1950)Pr!7. J. exf. Path., 31 540.
Rinaedini, L. M. — (1949) J. Endocrin., G, 54.

EXPLANATION OF PLATE

Fjg. la. — Tc-stes of n rnt fed nitrofurazone O-lo per cent in the diet for 25 days. Note
degenernf ion of f iiQ spcrmatogenic epithelium and the mucoid substance tilling many of the
spnce.s between distorted tubules. Hyperplasia of interstitial tissue is not apparent.
X23.

Fig. 111 . — Testes of a normal rate. x23.

Fig. 3o. — Atrophic seminal vesicle of a tumour-bearing rat. Note decrease in seminal fluid
and increase in fibro-musculnr stroma, x 7.

Fig. 36. — Seminal vesicle of tumour-bearing rat treated daily for 10 days with 10 mg. of
nitrofurazone intrnperitonenlly. The relative amounts of seminal secretion and fibre-
muscular gtroma resemble that of a normal gland. X7.

THE INELUENCE OF DIET ON THE ACTION OK TllIC
WALKER 256 CARCINO]\IA ON LIVER PliOTl-HN AND
NHJCLEIC ACJDS

CATHERINE M. CLARK and 0. A. .1. COODLAI)

From the Department of PhjsioloQtj and Btochetnisiri/. SI. Snlrnlor'.'i (tnllr^jr.
Univcr.silij of St. Andrnr.'i, Fife

Received for pulilicatioo XInrcli 1!). lOliO

• experimental tumour-bearing rats tliere is cvitleiicc t Iiat t lie growing t ninmir
interferes with the metabolism of protein in the tissues of Hie host (Stewart and
iiegg, 1953; ^son, Bernstein and Babson, 1954) and tliat lliere is a stam* in
tb^l” of f^'o liost sliows a gain in jirotein content while

tmourl'simik^o t^^ > foil). This elTect of a

in both fliat obtained by treating rats witli cortisone when an incre-isr

TrSikls^X T excretion is observed (Sillier and I’orter. 195:{ ■

arises as to wbet^^^ ond Munro, 1 959). 'J’ho (iiics' ion’

St mLht ^ of tumours on the metaboli^" of w

b?nSl tha?tL° r •'Adrenal cortex. It irimle'd

Begg, 1953) and in a S

that they may be hj^eractive presented evidence to suggest

tomodhySeSrfeoSn™^^^ and Munro, 195t)) diet was found

and ribonucleic acid (RKA) contL? ’”^'’^•'''^0 in jirotein

"^uencedbythelevelofproS L tbJ^ educed by cortisone was un-
degree on the calorific vMue of tb^. d t ^ ^ dependent to a considerable

content was not infiuenced bv tlo^ i ^ deoxyribonucleic acid (DNA)
dietarj^ conditions. ^ administration of cortisone under any of these

bearing rats iS; TTl ®^BceUular fractions of the w! of protein

directed at anv ^^fbough the action of the tum ° and turaour-

in the natten? T ^^e case of rSa thol ^PP-^ar to be

decrease in thp°l^™*®^^ distribution in the tumour b^^^ ^ significant alteration
bierease n t le °f P^tein found ProduciW

" iBi any alt of Wmour bearii ^’^iiotion. The

21 "™'>er of nuclei in the jit

328

CATHERINE M, CHAEK AND G.

A. J. GOODLAD

experimental methods

1 ™ overnight and those wei4m<r

160-200 g. were selected and lioused in individual metabolism cages ° “

limour moculaUon.~A specimen of Walker 256 carcinoml groTrn Mn-

' ‘T m dissected out and a suspensimi

of described bj' Tak^, Takano and Huggins (1952). I ml. portions

ol tins were injected nitrarauscularly into the right tliigh of each experimental
annual. Green, Benditt and Humphreys (1950) have demonstrated thatdietan-
protein level influences the growth of the Walker tumour during the induction
period but not subsequently and for this reason the rats were fed a diet of adequate
protein content for a preliminar}^ period of 6 days. At the end of tliis period
the tumours were easil 3 '’ palpable and rats having tumours of similar size were
selected to continue the experiment. A control series of rats from the same stock
were fed in parallel.

The first series of experiments was designed to stud}’^ the effect of dietan'
protein and calorie intake on the response of the liver to the presence of agroiving
tumour in the host. During the first 6 days (preliminary induction period) the
rats were fed a diet adequate in calories and protein content. Tliis was fed in two
parts. At 9 a.m., the rats received 1 g. of a vitamin-mineral roughage mixture
(IMunro, 1949) most of the calories being given in the form of 2-4 g. of starch
(potato) and 0*5 g. of glucose. At 5 p.m. a meal providing all the protein and
the remainder of the calories in the diet was fed, namely 2-4 g. of casein (un-
extracted grade, Glaxo Laboratories Ltd., Greenford, JEddlesex.), O-GO g. of
glucose and 0'42 g. of fat (margarine). For the remaining 5 daj's of the experi-
ment the calorific value of the diet was x’-aried by feeding in the morning 1 g- of
the xfitamin-mineral-roughage mixture either alone (low energy group) or together
xvitli 4 g. of starch and 1 g. of glucose (liigh energy group). The protein content
of the diet was varied b}'^ feeding in the evening either a meal containing 2-4 g. of
casein, 0'69 g. of glucose and 0'42 g. of fat (protein-containing group) or an iso-
caloric meal consisting of 1’89 g. of starch, 1'89 g. of glucose and 042 g. of fat
(protein-free group). In this way, the animals were divided into 8 groups,
comprising tumour-bearing or control animals receiving either a protein-containing
or a protein-free diet at a high or low calorie intake. On the sixth day ot this
regimen, the rats w^ere killed by stumiing and exsanguination.

In the experiments designed to study the distribution of BHA and pro
in the various subcellular fractions of liver and the mean DBA content ot liver
nuclei, the rats reeeix^ed the diet described above for the preliminarj^
period for the duration of the experiment (9-11 daj's from tumour inocii a i
that is all animals received an adequate intake of protein and ca ones. .
Analysis of liver.— The liver, spleen, adrenal glands and tumour were up .

were fo, p.0«« N by

Ribonucleic acid phosphorus (RNAP) and deo-vi^ibonucleic amd phosg^
(DNAP) xvere determined by the differential solubility of
iridiloroacetic acid by method previously described (MunroandHaismith,

Portions ofthe liver were taken for histological o 'L oILUit- fractions were

Intracellular distribution of RNAP and protein ^ content

prepared by differential centrifugation in 0-25 m sucrose and their HAA

LWEE COMPOSITION OF TUMOUE-BEARIKCI RAIS

32!)

determined as described by .Schneider (IMS). The irroleii, N eonlcnl. of the

different fractions was determined. , ,

DNA content of liver cell niicJei .-Nuclei were prepared bj citeic ac l
procedure, as modified by Smellie, Hnmpbrcy, Kay and Davulson ( Oo.,) J lie
OTclei suspended in 0-01 M citric acid, were counted in a hacmac\ tomctei . •
pSn of the suspension was also taken for DNAP deterinination (Miinro and
Naismith 1953) and deoxyribose estimation by the diplienylaniinc reaction
(Schneider, 1957). The DNAP content per nucleus was tlien expressed in juco-

grams (pg.).

RESULTS

The effect of various dietary conditions on liver, spleen and adrenal wciyhts and tumour
growth in rats bearing a Wallcer 256 carcinoma
The method of tumour implantation described by Talalay ct al. (1952) for (lie
standardisation of tumour grondh was emplojmd in the present work. Altbough
tumour size varied from experiment to experiment, it was relativel}’ constant,
within any one experiment. Tumour growth appeared to be independent, of
the various dietary treatments employed (Table I), a finding which is consistent

Table I. — Liver, Spleen and Adrenal Weights of Control and Tumour-bearing Rats under

Various Dieiary Conditions

Results are the means of 5 experiments ± S.E. Statistical analy.si.s
shows that the spleen and adrenal weights were significantly affected by
dietary protein (P < 0-05 and P < 0-01 respectively) ; that liver, spleen
and adrenal weights were significantly increased in the tumour-bearing
animals (P < 0-01 in all cases) ; that the increases in the spleen and
adrenal weights caused by the tumour were significantly greater in the rat.s
fed a protein-containing diet (P < 0-05 for interactions between tumour
and dietary protein intake) ; that the magnitude of the increase in liver
weight was not significantly affected by diet (P < 0-05 for interaction of
tumour wth dietary protein and calorie intakes) ; that tumour weight
was not significantly affected by dietarj’^ protein or calorie intake (P > 0-05
m both cases for interaction). '

Dietary

protein

Dietarj-

calorie'

value

Tumour

weight

(g-l

.Vone

/Low .
\High .

ll-4±2-8

9-3+2-2

-Vdeqvmto

/Ix)W .

\High .

10-5±2-0

n-3±2-4

Liver weight
(g./lOO g. of initial
body weight)

, - A

Tumovur-
Control bearing

series series

2-78±0-30 3-14+0-34
2-81±0-15 3-6I±0-13

2- 64±0-14

3- 20i0-15

3- 91+0-35

4- 22±0-68

Adrenal weight
(mg./lOO g. of initial
body weight)

Tumour-
Control bearing

series 6crics°

31-4±1-4

29-4±4-3

41-04:4-9 .
39-8±4-G .

31- 0±3-6 48-2-f-C-l

32- 0±2-0 49-4i8-7

Spleen weight
(mg./lOO g. of initial
body weight)

Control

series

I’umoiir-
bearing J
serie.s

348+02 420+84
353±41 .")53il02

393±70 821-1-142
374±12 813±133

330

CATHERINE M. CLARK AND G. A. J. GOODLAD

M'ciglits of these organs were observed under all dietary conditions (Table II
T he ga,n m hver Mv.ght caused by the tumour was not significantly Lfluelced'
j diet.. T he increases in spleen and adrenal weights in tumour-bearino animals
marked in the animals receiving a protein-contaLing diet

effect of the WaU:cr 256 carcinoma on the protein and michic acid content of ml
liver

Stewart and Begg (1933) found that the Walker 256 carcinoma produced liver
protein dcjiosition in rats fed a high protein diet. In the present study an increase
in liver protein content was observ’ed in tumour-bearing rats, irrespective of
tlie level of protein and energj^ fed (Table II). Tliis increase in liver protein was

Tahle II, — ud Comparison of the Protein N, SNAP and DNAP Content oJlheLim/(
Tumour-bearing and Control Bats under Various Dietary Conditions

Rc.sult.s arc the means of 6 experiments d; S.E. Analj'sis of variance
shoir.s that the tumour caused significant increases in liver protein, ENA
and DNA content (P < 0-01 in all eases) and that these effects are inde-
pendent of dietary protein and calorie intake (P > 0-05 for all interactions).

Dietary

protein

None

Adequate

Diefarj’

calorie

value

{

Low .

High .

Low .
High .

Liver protein N
(mg./IOO g. of initial
body weight)

Mean

per-

cent-

Tumour- age
Control bearing dif-
scries series ference
57-3±5-0 C5-J±5-4 +14
53-G±G-0 GS-g±5-7 +28

57-4±0-2 S5-9+9-4 +50
72-0±S-l SG-9±I7-9 +21

Liver RNAP
(mg./IOO g. of initial
body weight)

A - - -

Mean

per-

cent-

Tumour- age
Control bearing dif-
series series ference
2-71±0-19 3-64±0-14 +34
2-63+ 0-14 3-90±0-34 +52

2- GS±0-23 4-37±0-ol +63

3 - 07 ± 0-22 4-49±0-S9 +46

LiverDX.tr
(mg./IOO g. of iniM!
body weight) __

' "Ik-

f'-'

«:•

Tumour
Control iMrin!

series rvrics tr-

1-07±0-U MSiO'S'i:
1-04+0-09 M9±0 (d'i

1-OS+O-lO

also accompanied by increases in both liver BNA and DNA, protein and ca ones
intake again having no significant effect on the magnitude of these c laii;, =
(Table II). .

In each dietary group the magnitude of the increase in '^rrrntl

(34-63 per cent) was much greater than that in either liver protein (1 o pe
or DNA (10-19 per cent).

Comparison of the intracelhdar distribution of RNA and protein in the Ui^ /
tumour-bearing and control rats ^

Since the Walker 256 carcinoma produced an increase in both
protein content a study of the intracellular distribution of EiS < .Itcmpt to
the livers of tumour-bearing and control rats was 5 'factions

ascertain whether tliese increases were due to changes in any p^ ‘
of the liver cell. In the preceding series of experiments it
increases in ENA and protein were independent of the dietary

lwer composition op tumour-bearing rats

;i:!i

Consequently in the present study only tumour-bean g

an adequate protein and calorie intake vere employed, fal, e III » " j.

although there was a considerable increase m tlie amount of RlsA
tumour-bearing aninals (P < 0-1), the percentage di^nbution of
different subceUular fractions was not significantly different from that fonn 1
the normal rat liver.

Table III. — The Intracellular Distribution of BNA P and Protein N
in the Livers of Control and Tumour-bearing Rats

The results are the means of 4 experiments ± S-E; hi the case of RN.XP
and 3 experiments ± S.E. in the case of protein N. Stati.stical anahvis
(“ t ” test) .shows that the tumour caused a significant increase in total
liver RNA content (P < 0-01) ; that there was no significant alteration
in the pattern of RNA distribution in the subcellular fractions of the
livers of tumour-bearing rats ; that there was a significant fall in the
proportion of protein N present in the mitochondrial fraction of the livens
of tumour-bearing animals (P < 0-05).

Total amount
per liver
(rag./lOO g.
of initial

Treatment body weight)
RNAP— . . 3-01±0-03

Control

Tumour-bearing . 5-64±0-01

Protein N — . 74-5±0-7

Control

Tumour-bearing . 88'3±G'7

Distribution in the subcellular fractions
(expressed as a percentage of the whole homogenate)

I ' >

Mito- Micro-

Nuclear cbondrial somal Soluble Recovorv

7-6±0-9 13-5±l-4 49-C±2-0 2d-I±l'9 9.7-8±2‘3

9-l±0-9 n-5±3-3 45-7±2-8 2S-7±r,-5 n5'0±2-8

15-9±0-3 28-6±2-8 18-C±3-0 35-C±3-4 OS-Oif)-'.

20'5±3-8 13-7±3-6 19-9±3-3 30-9±4-4 Ol-Oil-S

The protein data (Table III) suggest that there was again an increa.se in the
protein content of the livers of tumour-bearing animals. However due to the
variation between animals this increase did not attain statistical significance.
Nevertheless the pattern of distribution of protein in the different subcellular
fractions was significantly altered by the presence of a tumour, namely by a fall
"p protein present in the mitochondrial fraction

Mean DNA content of nuclei isolated from the livers of control and tumour-bearing

. i"crcns=cl,

m the mean nuclear DHAP. The dietarv increase

“in f bTth by XTphorusTe!

-lation of DHA and by the diphenylamine

a,<, „« Jiff., obtiedln'’ U'i

332

CATHERINE M. CLARK AND G. A. J. GOODLAD

Table Comparison of the Mean DNAP Content of

Liver Cell Nuclei in Control and Tumour-bearing Bats

experiments ±S.E. Statistical analysis
♦ + f 1- ^’^ere was a significant increase in the DNAP con-

inean°DNAP en alteration in the

mean DAAF content of the nucleus.

Treatment

Kono

Tumour

weight

(g^l

9-0i:0-8

Lirer DNAP
(mg./lOO g.

of initial
body weight)

0- 97±0-02

1- 20i:0-06

DKAP (pg./nuclens)
as determined by

, ^

Phosphorus Beoxypentose
estimation estimation

0-96±0-02 O-DOAO-OS
0-95±0-04 0-90+0-03

Histological examination of the livers of control and tumour-bearing rats

Histological examination of the livers of tumour-bearing rats failed to show
any evidence of centres of haematopoiesis such as have been described by Ventura.
Richer and Sel}^ (1957) in livers of rats bearing large Walker tumours (ca. 40 g.).

DISCUSSION

In the present series of experiments it has been found that the Walker 2a6
carcinoma causes an increase in the weights of the liver, spleen and adrenal glands
of the host. The increase in liver size was independent of the level of protein
and calorific value of tlie diet wdiile the increases in spleen and adrenal weights were
more marked in rats receiving a protein-containing diet. Tumour growdli also
appeared to be independent of the dietary conditions employed.

Examination of liver composition showed that there was an increase in the total
amounts of protein, RNA and DNA in the livers of tumour-bearing rats, irrespec-
tive of the protein and calorie content of the diet. The fact that even the livers
of tumour-bearing rats receiving a protein-free diet showed this increase suggests
that the protein laid dowm in the liver arises from the breakdown of carcass protein
wdu'ch is linowm to occur in tumour-bearing animals (Sherman, Morton and Slider,
1950 ; Regg and Dickinson, 1951). It is of interest to note that Green et al.
(1950) could not demonstrate any increase in the protein content of the livers o
rats bearing the Walker 256 carcinoma yvhen these animals had been extenshe!)
depleted of bodj’^ protein by feeding them a protein-free diet for three wee s

before tumour inoculation. j i r n

Tins action of the tumour in causing an increase in liver protein by dep c nic
the carcass appears similar to the action of cortisone. There is evidence t la ii»
hormone acts primaril}’’ on the carcass causing a breakdown of tissue
resulting in an increased supply of amino acids to the liver (Goodlad an i u -
1959). Due to the sensitivity of the growth of the Walker 256 ‘ j

dietarj’^ protein (Green ef al., 1950) the dietary conditions emplo 3 Td m le P ‘
work wmre not exactly similar to those of a preceding studj^ of the ac ion
on the response of the liver to cortisone adminstration (Goomad and i unr , >
Nevertheless there would appear to be certain differences in the respons .
liver to these two circumstances wliich are not related to the change ‘
mental conditions. In the first place, it was found that the deposi lo
protein and RNA caused by cortisone was related to tlie calorie m a • ,
the increase of these two constituents in the livers of tumour-bearing

lwer coj^iposition of tumour-bearing rats

treated?ats. These results therefore suggest that the ohscncvl

composition in tumour-bearing animals may be brought aliout bj a

mechanism from that by wliich cortisone exerts Its effect.

Studies on the intracellular distribution of RNA and protein showed that, u Ink,
the PvNA of all subcellular fractions was increased by the action of the tumom,
the percentage of the total liver protein present in the mitocliondnal raction
was lowered in tumour-bearing animals. Whether this was due to a decrc.isc
in the amount of protein per mitochondrion or to a decrease in the number of
mitchondria relative to the other cellular components cannot be decided from t he
data available. Allard, de Lamirande and Gantero (1953) have in fact found t hat
the mitochondrial population (expressed as mitochondria per cell) of liver cells
adjacent to a growing hepatoma but themselves free of tumour was significantly
lower than that of normal liver cells.

The total DNA content of the livers of tumour-bearing rats was significantly
increased. A rise in liver DNA may be attributed to one of the following : —

(a) Centres of extramedullary haematopoiesis may be present. These have
been observed in the liver and other organs of tumour-bearing animals (Bcgg,
1958).

(b) The degree of polyploidy of liver nuclei may have increased. Lcuchtcn-
berger, Leuchtenberger and Myeki (1958), working with mice, showed that the
amount of DNA per liver nucleus increased folloing injection of tumour DNA.

Thomson, Heagy, Hutchison and Davidson (1953) have investigated the mean
DNA content per nucleus of cells isolated from different tissues of tlie rat. They
found that, while the mean DNAP content of liver nuclei was of the order of
0'90 pg. per nucleus or greater, that of spleen and bone marrow tissues, where active
haematopoiesis occurs, was only 0-63 pg. and 0-67 pg. per nucleus respectively
Leucocyte nuclei had a mean DNAP content of 0-64 pg per nucleus Tims t f.,II
m the ™e.n DNA content of liver cell nuclei night b”“erd Tt inffl aM™ J
haematopoietic centres was occurring to any great extent An increa“ nolv

formation of new t c/r T^^^^vi^r to^ie

that resting hver mitosis rates in rats bearing tlSValkS’Sfi

seven tunes greater than normal (Hemingway I960). ^ carcinoma ivere

1 nti rr SUMMARY

1 . Jue effect of the WalFor

•stniliecyn rats receiving diets vaW^p'rXfrand oT""

- rumour growth was found to L . ^.nd calone content,

meitte emplojecl. *•' “Apendent of the various dietar,- treat-

334

OATHERIKE M. CLARK AND G. A. J. GOODLAD

and calorie content of tlie diet in the case of liver but the rises in spleen and adrenal
Aveights observed in tumour-bearing animals u'ere more marked in rats receiving
a protein-contammg diet. °

_ 4. An increase in the protein, RNA and DNA content in the liver vas observed

m tuinour-bearmg rats, under all dietarj^ conditions. The percentage increase in
IvJN A being much greater than that of either protein or DNA.

5. The intracellular distribution of RNA in liver was unaffected bj^ the presence
of a tumour. However protein distribution was altered in the livers of tiiinonr-
bearing rats, there being a fall in the proportion of protein found in the mito-
chondrial fraction.

0. The mean DNA content of liver nuclei in rats bearing the Walker carcinoma
did not differ from tha.t observed in normal rat liver.

Idle authors wish to express their gratitude to Dr. H. N. Miinro of the Depart
ment of Biochemistry^ Glasgow University^ for valuable advice and discussion,
Professor A. Haddow F.R.S. of the Chester Beatty'^ Research Institute, London,
for supjilydng a suitable strain of rats and samples of the Walker 256 carcinonnr
and Dr. W. \V. Park of the Department of Pathology, St. Andrew’s University, for
advice on certain histological points. One of the authors (G.A. J.G.) carried out
part of this work during the tenure of a Beit Memorial Fellowship at the Depart-
ment of Biochcmistiy, Glasgow University, and would like to thank Professor
J, N. Davidson, F.R.S. , for facilities granted. The latter stages of this work rvere
supported by* a grant from the Medical Research Council for expenses and scientific
assistance to oiro of us (G. A. J. 6.) for which we are most grateful.

REFERENCES

Anmum, C., de Lajiirande, G. and Cantero, A. — (1953) Canad. J. wed. Sci., 31, 103.
Aletsok. j. B., Bernstein, E. H. and Babson, A. L. — (1954) Fed. Proc., 13, 174.
Begg, R. W. — (195S) Advanc. Cancer Bes., 5, 1 .

Idem, AND Dickinson, T. E. — (1951) Cancer Bes., 11, 409.

Goodlad, G. a. j. and Munro, H. N.— (1959) Biochem. J., 73, 343.

Green, J. W., Benditt, E. P. and Humphreys, E. jM. — (1950) Cancer Bes., ID,
Hemingway, S. T. — (1900) Nature, Bond., 185, 106. ,

Leuchtenberger, C., Leuchtenberger, R. and Myeki, E. (I9oS) rroc. m. -
Sci., Wash. 44, 700.

Munro, H. N.— (1949) J. Nnlr., 39, 375.

/dew? AND Naisjhth, D. J.— (1953) Rioc/few. .7., 54, 191.

SemnDT, G. and Thannhausee, S. J. — (1945) J. biol. Cnem., 161, 53.

Schneider, W. C. — (194S) Ibid., 176, 259. S P and

/rfe???— (1957) in “ Methods in Enzymology ”, vol. 3, p- 680, Ed. by- Colo

Kaplan, N. 0., New York (Acad. Press Inc.). ,

Sherman, C. D., Morton, J. J. and Midee, G. ^ ’ ’ ■ ’

SiLBEE, R. H. and Porteb, C. 0.— (1953) Endomrwhgy, 52. 51^- X_-(l!}5.7)

Smellie, R. M. S., Humphrey, G. F., ILay, E. R. M. and Da\u * ,

Biochem. J., 60, 177. „„

Stewart, A. G. and Begg, R. W.-(i953) Cancer J3, 556 5b0
Talaeay, P., Takano, G. M. V. and Huggins, C--(I952) /i?d 834. ^

Thomson, R. Y., Heagy, F. C., Hutchison, W. C. and Daitdso. ,

Biochem. J., 53, 460. „ . p g 279 .

Tremolieees, J., Dekache, R. AND Lowy, R. 17’ ‘>15

Ventura, J., Richer, C-L. and Selye, H.-(1957) Cancer Bes. 17, Ao.

THE IN VITRO
ISO-AHTISEEA ON

CYTOTOXIC activity OF MUmXK
NOIIi\IAL and JIALICtNANI CELLb

PATEICK O’GOBMAXt

Fr.m tU Devarlmeni of Expermenlal Pathology. Guy'.s Hospital Mcdkal School
Received for publication April 13, 1000

Tv mice passive immunity to leukaemic homografts has been obtained hy
transfers iso^ntibodies against both the H-2 antigens and the

fo Sled X-^e antigensrexamples of which have been found m several leukoses
(Amos and Day, 1957 ; Gorer and Amos, 1956 : Gorer, personal commumcation).
Histologically, leukaemic grafts can be seen to be destroyed by a massive cMida-
tion of fluid into the graft on the fourth and fifth day ; thereafter host cells appeal
vhich act only as scavengers (Gorer, 1958). Sarcomatous grafts, on the other
hand, are invaded by a synojdium of host cells which engulf and destroy t lio graft.
The antigens of the H-2 system are the major antigenic factors controlling tlic
fate of homografts in mice, and are determined by a long series of alleles on the
ninth chromosome. Present knowledge of tliis system has been reviewed by
Gorer and Mikulska (1959).

These results suggested that iso-antibodies play an important part in homo-
graft rejection, particularly of leukotic grafts, and might have a detectable effect
in vitro. There have been few purely in vitro studies of this effect. ^Miller ( 1 956)
failed to show any effect upon cells growing in tissue culture of e.vxiosure to iso-
antibody ; the cells were of epithelial origin and the presence of active complement
was not ensured, ilouse serum is usually devoid of complementarj' activity
in vitro, and Eitz (1911) stated that mouse serum was deficient in the “ end-piece ”
of complement, whilst Broum (1943) believed it to lack the second component,
C'2. IMcGhee (1952) demonstrated murine complement in vitro and convincing
evidence of its activity in vivo has been provided by the experiments ulth diffusion
chambers reported by Algire, Weaver and Prelin (1957), who obtained hvsis of
Hcla cells and by Amos and Wakefield (1958) who demonstrated lysis of niouse
leukotic cells.

Sebrek (1936) has described a method of distingui.shing viable and non-
yiable cells in a suspension by means of differential staining, using eosin, methylene
bhie and t^Tian blue, dj^es wliich stain non-viable cells, whilst viable cells are
not affected, as Pappenheuner (1917) had shoum many years before. A technique
for stiidjmg the effect of murine iso-antisera in vitro was briefly reported \v

by tbe TJnivei.ity of London for the degree of

+ Trcent eddres., ; Group Laborato^-, Lewisham Hospital, London, S.E.13.

PATBICK O’goRMAN

leucocj^es and upon several leukoses =

3IATESIALS

from the inbred lines Strain A, 'BALBIc, G,Hand
Co/Bl, maintained in the lab^oratory of Dr. P. A. Gorer, and also the progeny of
the backcross (A X C57B1) F, x C57B1. ^ ^

Gowplewew/..— Guinea-pig complement was used, obtained by cardiac puncture
and stored at —20° C. until needed.

/■D — ^"'0 ascites leukoses were used, EL4 (057B1 strain) and CL:’

(BALB/c). EL4 has been maintained by transplantation for many years, and
^riii not grow subcutaneously in foreign strains, but sometimes 'tvill do so intra-
peritoneally. CL2 is of recent origin and is strain specific. Two other leukoses
CLl (BALB/c) and ELS (C57B1) are of recent origin and are strain specific, but
produce generalized leukoses m'th massive enlargement of the spleen. Tlnre
ascites sarcomata u-eie used, BPS (CgH strain) and Sal and MTC lA (both .i
strain). All of these tumours sometimes grow progressively'’ if inoculated intra-
peritoneally in large closes into foreign strains. In moderate dosage, sub-
cutaneously’’, they’’ are strain specific. Tlmee other strain specific tumours vero
used — the mammary’- carcinomata EMT (C57B1), AIMT (A strain) and CTl
(BALB/c) and the leukosis ELS ; all were maintained as sohd subcutaneous
grou’tlis.

Iso-an(isera . — Details of the hyper-immune iso-antisera used will be given at
the appropriate places in the text.

Dyes. — Both eosin and try'pan blue were used at a concentration of 1/2000 in
Tyrode’s solution, filtered after preparation. No significant difference was obser-
ved in the results of parallel experiments using the two dy'es, but trypan blue
gives more clear-cut staining.

METHODS

Prefaration of cell suspensions

1. From ascites himours. — The ascites was diluted in Ringer’s solution m
the case of the non-haemorrhagic leukotic ascites, and in 3 per cent sonum
citrate in the case of the haemorrhagic sarcomatous ascites. Total cell couin
ivere made at once, and the dilution adjusted vith normal mouse serum an^
Ringer’s solution to give a final count of approximately' 30,000/inm. m •
normal mouse serum. Yiabifity counts gave from 0-5 per cent stained ce s
the case of the ascites leukoses, and about 0-10 per cent in the ascites ‘ .jj,

2, From spleen. — ^The same method -was used for both normal ana e
spleens. The animal was killed udth chloroform, the abdomen opene , m sp
exposed and the splenic artery and vein were cut vnth fine scissors.
syringe was fitted ivith a fine intra-dermal (No. 26 G.) needle an

sterile Ringer’s solution. The point of the needle was introduced u ^
capsule of the spleen and light pressure applied to the plunger, imm '
small white area appeared around the needle point, which extende as . ,
was maintained. After about 1 ml. had been injected the needle nas
and reinserted in a different position. In this way the wdiole spleen ufc ‘
perfused. If the splenic vessels are not divided, the liver an 'i J

CYTOTOXIC ACTIVITY OF MUHIKE IRO-AKTISKUA

IVM

perfced ,t to same toe. and Me v V, rr;;.r,U,,e

these organs than by cararalatoi of «■» »*n " .

injection. After perfusion the \ sen,,,,. S,„„n

mhrced „*h smaU scissors and suspe, deil m ' ,.e„,„i,„.,l

approtoately 30,000 lencoej-tcsjnnn.a Viability counts usually ahoued „ >„„t

5-10 per cent stained cells.

Titration of comphment

Undiluted gninea-pig serum was often toxic to the target cells "h. s^l its
complementary activity declined rapidly on dilution. It was tliercforo titratr
in terms of its ability to complement the cjdotoxic activity of iso-.mit^sera .A
serum knovm to be toxic for the target cells was used at a dilution ofl : o aiul I lie
complement was titrated in the dilution range 1 : 1 to 1 ; 4 ; it was seldom active
at greater dilutions. Controls of normal serum and another iso-antisenim known
not to be toxic for the target cells were used.

Antibody titration

The cytotoxic activity of an antiserum was usuallj' investigated in the form
of a titration, to exclude 2 oning.

Doubling dilutions of each antiserum to be tested were made in 1 ; normal
mouse serum. Control tubes, containing only the diluted normal serum, were
included. A volume (0*05 ml.) of cell suspension was added to each tube and
finally a volume of diluted complement. The rack was incubated at 37° C. for
45-75 minutes, except in the experiments on the timing of the reaction. After
incubation dye was immediately added to all the tubes to make a final dilution of
1 ; 10. The tubes were left for a few minutes before counting to ensure correct
staining.

Two methods of reading the tiwation were employed. In the first, differential
counts were made on each tube. In the second, the two control tubes were counted
and the cells from the rest of the row were streaked across a glass slide and
inspected, ^e results were expressed as -f ++, -f -u etc., the terra AC being
used when the effect was almost complete and there were virtual^ no viable cells
to be seen; a negative was scored when the number of stained cells approached
that m the control tubes Tliis method is entirely satisfactory^ for tlm routine
titration of a seinm,but the method expressing the result a,s a percentage viable
count was found to be more delicate when comparative work was beim done
Results obtained by the two methods are strictly comparable. °

namayghttination

In vivo absorption

33S

PATRICK O’goRMAN

Lr;o!-r;l' r xl. ^ ‘"

RESULTS

The morphology of the damaged cells

AVI considerable variation in the appearance of the stained celh

Win St some retained a normal rounded outline, in AAdiich the nucleus could
barely be seen, other cells became sn-ollen, sometimes grossljr so. The outline of
these cells became irregular and indistinct and occasional^ the cell memhranc
appeared to have developed bubbles. In the more severely affected cells the
micieus could be clearty seen M'heu trj'pan blue was used as the stain. Actual
lyvsis of the cells was not seen.

Effect on 'normal and malignant cells

Eo significant difference could be detected betAveen the results obtained using
normal or leukotic AA’hite cells, but ascites leukoses Avere preferable as the suspen-
sions contained less debris and fewer stained cells than those made from spleen or
sarcomatous ascites.

The effect of heat

Heating the antiserum at 56° C. for 30 minutes aa'ss without effect on thetitre.
Jiaie of reaction

jMiirine iso-antisera appear to be toxic to homologous cells even after very
short exposure, in the presence of active complement. In an experiment m
AA’liicli BALE /c anti'BPS Avas tested on normal CjH leucocjffes, 90 per cent of the
target cells Avere stained after only 10 minutes’ incubation at 37° C. The contra
at this stage contained only 5 per cent non-Auable cells. After 20 niinutes
incubation 95 per cent of the cells AA'ere dead ; the increase is probablj noy
significant. On the other hand, prolonged incubation, i.e. for more tian ■,
hours produced high numbers of dead cells in the control tubes.

Tifne of appearance of cytotoxic antibody

Groups of four mice Avere bled on successive daj'^s after inoculation a\
suspension. The blood from the individuals of each group was poo ®
cjdotoxic aetiAuty of the serum tested on appropriate cells. The haema^jg

ing titre Avas determined at the same time- i • u ■ irv I The

The results of one such experiment are expressed graphicahy ^
animals Avere BAIjB/c, inoculated subcutaneously udth • '

tested for cytotoxicity on EL4, and for haemagglutinating ac ia i j i j,gn,a(7.
C57B1 red ceUs. As can be seen from the graphs neither
glutinin was present on the eighth day after inoculation. o i pp
ninth day, and the titres rose together on the same day.

CYTOTOXIC ACTIVITY OF MURIXK ISO-ANTISKUA

The Nature of the Antigenic Siistcw

using the method of in vivo absorption described by Amos (lA..,). Oiu .m

BALB/c ANTI EL4 TESTED

E L 4 AND A A N D C 5 V I) I H 11 C ' r.

z ““bY Y 5- mmTc t ict

and the sera from each’iouTnL ntw 24 hours later

normal mouse serum. BALB/c antiElA so EL4, m parallel with

anti-E and anti-B^ and these

S lould be absorbed, however bv Dassf^el P-Sf ’'y Passage in BALB/c. They
<>'<1 occur. When tested b£ha\QSa&^^ *hi.; in fac't

og urination the passaged serum gave a

340

PATRICK o’GORMAN

Tahlk I.-

BALR/c

nnti-Et,4

pnssngod

BALB/c
(C57B1 X A)F,

-Absorption in vivo of Cytotoxic Antibody

Percentage non-vi'able cells at
antiserum dilutions of ;

, — »

1/1

1/2

1/4

100

1/8

100

Haemagglutinin

litres

( *

A strain C57B1
1 : 4096 1 :64
I : 256 0

fx p tj n Vr'^® ^ “ {which would detect anti-E) and no litre

Tj \ T ® anti-E) nor for C57B1 cells {anti-E’’ and anti-E) whilst the

liALBIc passaged serum gave litres of 1 : 4096 for A cells, 1 : 512 for C3H
fo<’ C57B1. As can be seen from the table, the serum passaged in the
(Lo/lil X A) Fj showed no residual c3rtotoxic actiidty for E14 tumour cells killing
a inaxinnnn of onlj^ S per cent. The serum passaged in BALB/c on the other
hand lulled 100 per cent of the target cells. The C3rtotoxic antibody had been
absorbed out in a similar manner to the haemagglutinating antibody.

Sera of this type (BALB/c anti-EL4) when tested in vivo, can be shorni to
contain anti-X. This antibodj:' is not absorbed when the serum is passaged in
Go7Bl or Co7B! Fj, and should therefore have been detected in this experiment, if
it was in fact the antibod3^ concerned. Even a short absorption left no detectable
C3’■totoxicit3^ 0-2 ml. of BALB/c anti-EL4 was injected into BALB/o and
(C57B1 X A) Fj, and the animals were bled out after only two hours. The
C3d;oto.\’io and haemagglutinating antibodies avere both absorbed b3^ the (Co/Bi
X A) Fj, tiiere being no residual activity, although the BALB/c passaged serum
lulled 80 per cent of the target cells and gave a haemagglutinin litre of i
for A cells. The difference betaa'een the titre of the absorbed serum in this experi-
ment and that shown in Table I is due to the different amounts of serum given
(0-2 ml. and 1-0 ml. respecthml3:^}.

These results suggested that both cytotoxic and haemagglutinating antibod.v
were determined by the same antigenic system, in other words the H-2 system,
and a series of experiments was undertaken to prove this theory.

Bacl'cross experiments

A cross was made between A X C57B1 and the Fj was backcrossed to
In these crosses some of the H-2 genes are segregating, and 50 per cent 0 m
progeny of the backcross vdll have the A strain genotype. The A strain m
the H-2 constitution CDEFHKRA’VTfZ whilst the C57BI can be j^tten as
D'’EFK'’HVZ (Gorer and Blikulska, 1959). Restricting the discussion to 1
pertinent genes, the (A X C57B1) F^ is CDK/D’’!!’’ and the B backcross
D'’K’’/D'’K‘’ or OEK/D'’K’’. Tissues of the animals carrying CDK will nauu. .

absorb the appropriate antibodies from sera. onHnens

The progeny of the B backcross were tested for the presence ol these am
by haemagglntination and divided into H-2 positive (i.e. carrying G 1
negative (lacking CDK). Equal numbers of H-2 positive jvU

were tested by the technique of in vivo absorption (Amos, Ltoo).
an intra-peritoneal injection of 0-1 or 0-2 ml of C57BI anti-MTC
an A strain sarcoma and this serum contained anti-C, anti-D , yr\

The mice were bled after IS hours and the sera tested for . pf

excellent fit was obtained with the results of typing by haemagg u

CYTOTOXIC ACTIVITY OF MUBIXE ISO-AXTTSEHA

•in

12 B backcross mice 6 were H-2 positive wliilst

iLro^aU

No discrepancies were found.

(H-2a anH.H-.U, on,. „0 nl.o.,, „.
„o„tta'l.aemaggl«tM.s ooly erratically b„t are

sometimes for the CgH sarcoma BPS. A sample of H-2a nnti-H--l. v
prepared by Dr. George Snell in the I-R strains AlvK and AKll
hii^ These strains differ only at their H-2 sites, and so any iinji
must be against an H-2 component. This serum was also toxic for C,H cucoc\ tcs.
killing 74 per cent, as against 4 per cent in the control. A sample ol 1 l-'-n ant i-
H-2k prepared in this laboratorji^ killed 90 per cent of the leukotic cells. AOit her
serum was toxic for BALB/c or C57B1 leucocytes. Gorer and Mikiilska {19.79)
quote evidence from their crossover combinations to show that thi.s new antigen
should be on the D part of the chromosome and propose the tentative label 1)'^.

Anti-K'^.—The antigenicity of the alleles of K was investigated using the
crossover combinations described by Gorer (1956). An antiserum was ]n-ci>are(l
in the D— K+ (H-2h) crossover against the C57BI (H-2h) tumour EL4. It was
found to be toxic for cells from C57B1 and the D+K— (H-2i) crossover, but was
not toxic for the B+E— (H-2g) crossover. The D+K— cros.sovcr could only
have acquired its antigen in the K position from H-2b, whilst the 13+E— cross-
over could have acqrured this antigen from either H-2b or H-2d. If it had come
from H-2b then this serum should have been toxic for the B+E— cells. That,
the reverse happened shows that there must be two different antigens in the K
position on H-2b and H-2d, and the newly identified antigen on H-2b has hcon
termed K*’. The effect could not be due to an antigen in the C position, for it
could only have come from H-2b and the serum would therefore have been toxic
for B+E— cells.

The Susceptibility of Leukoses and Sarcomata to Cytotoxic Antibody
A. The leukoses

Eli -\cpr many experiments were performed in which EL4 cells were used

S ion f wrf u susceptible to antibody, and tj^ically

90-100 per cent of the EL4 cells are killed against 1-5 per cent in the control

ascites leukosis. In an e^pertment hTSdr^CLr^ enlargement, and CL2 is an

with BALB/c anti-EL4 and A aLi-CTl ti?^ f 0 P^'^^'lel

Die B.-Udl/c anti-EL4 lulled 100 per cent EL4 SkTf «’^tained.

on he other hand, the anti-CTl kffled iSv 2 utr c^t W ^

Both sera were used at a dilution of 1 • l Tn <^^1-

• another experiment the BALB/c

342

PATRICK. 0 GORMAK

BALbTc anc7A?ntfrTf' ^ anti-ELt, C57B] anti-

HT 1 V I r, 1- ^ anti-EL4 showed some slight toxic activity aoainst

GLl, for at a dilution of 1 : 2 it killed 20 per cent of the cells, compared 10
per cent in tlm control tubes ; differences of this order may not be significant.
The two anti-B^B/c sera produced 75 and 73 per cent stained cells respectively
tliere was no difference in the non-idable cell counts in the A
anti-EL4 and control tubes. This leukosis if anjdhing was slightly more suscep-
tible to the cjdotoxic effect of tlie anti-BALB/c sera, the figures being 81 and 8G
per cent in the two cases.

B. The sarcomata

Details of the two sarcomata BPS (CjH) and Sal {A strain) have been given
above. There was a strildng difference betn^een the results obtained with the
two tumours.

BPS . — Most of the experiments on BPS used samples of BALB/c anti-BPS,
but A anti-BPS was also included ; sera of the latter type contain anti-P'‘ am!
have been discussed above. The BPS tumour cells are not as susceptible as
leukotic or normal leucocjdes to the action of antibody ; in most e.xperiments non-
viable cell counts were of the order of 40 to 60 per cent at the lower dilutions of
antibodj' and on only one occasion did the count e.\‘ceed 70 per cent. The
difference between the susceptibilities of BPS and normal C3H leucocjdes is shorn
in Table II, which illustrates an experiment in which both were tested with A

Table II. — Cytotoxic Effect on BPS and Normal Lmkcocyles

Percentage non-viable cells at antisenim* dilutions of:

Target cells

Controls

1 : 2

1 : 4

1:8

1 : 16 1 : 32

BPS .

23 21

52 30

CH 3 leucocytes

14 a

93 81

* Antiserum was A anti-BPS — see text.

anti-BPS. AA^ien tested wdtli B ALB /c anti-BPS in parallel with EL4, BPS showed
less than 45 per cent stained cells, whereas the leukosis showed 95 per ceid. ^
Sal . — Sarcoma 1 appears to be completely insensitive to the in vitro cjdo-oxi
actixdty of iso-antisera, and despite repeated testing, with different ”
convincing evidence was obtained of any toxic effect. A strain leucoc) ^
as fully sensitive to antibody as are the leucocjdes of anj'' other strain. •
III shows the results of tests made in parallel on Sal and A strain eucocj

Anti-serum

C,H f
anti-AJIT \

CoTBl . /

anti-MTC \

BALB/c /
anti-BPS \

Table III.— -Dytoteic Effect on Sarcoma 1

Percentage non-viable cells at antiserum
dilutions of ;

Controls 1 : 2

1 S 5

8 6 90

-i

1 : 4

1:8

1 : 10

Target cclL
Sal

A strain

leucocytes

Sal

A strain

lcucoc.vt«

Sal

A strain

leucocytes

CYTOTOXIC ACm-ITY OF m'BlXF- ISO-AXTISl®.'

three different sera aU of which contained BALi^'p'd-'-lu’S

aAVT and MTC are botli A strain strain leiicocvtcs.

rontains anti-E and anti-K “[ "J"* was (ostod on n'll. of one

lanS*? “e“r»r— E-n in Tabic IV. and ..rov.dc s.r.U.m

Table W.-Cytofoxic Effect upon Three Tumours

...-a*

Percentage non-vinble coUit at antincnuu
dilutions of ••

Target cells
EL4 .
BPS .
Sal

Controls
16 12

1 6

3 ■>

1 ;2
ns
61

1 ; -1
9S
49

1:8 1 : lf>

95 91

56 61

3 9

Tt «T Ofaw.

evidence of the difference in susceptibility to antibody of tlic cells of the thriT
tumours.

DISCUSSION

The function of circulating antibody in homograft rejection b^s been t he .subject
of much discussion. By technical modifications of Gorer and ;Mikul,ska s 0 ••;> *)
method H-2 antibodies have now been detected in mice before the histological
onset of the homograft reaction (Gorer, Mikulska and O’Gormaii, IDoD) and the
demonstration in the present work that these antibodies have a cytotoxic effect
on leukoses and sarcomata is evidence against a purely cellular mechanism. 'J'he
cytotoxic technique is not as sensitive in antibody detection as is the huinari
serum ; dextran method, but has been the means of idcntifmng two “ new ”
H-2 antigens, K'’ and D

Ho evidence was found of any in vitro cytotoxic effect of the anti-X described
by Gorer and Amos (1956). This may be because anti-X is not toxic under these
experimental conditions, or because anti-X is not a c}i:otoxic but a cytostatic
antibodjq holding the graft in check until the body’s defences complete the
rejection.

Circulating antibody plaj's a much more decisive role in the destruction of
leukotic grafts than in any others, and for this reason it would seem that leukotic
cells or normal white cells would be the material of choice for the investigation
of the action of antibody. It is unfortunate that Algire and his co-workers did not
include such cells among the targets which they placed in their diffusion chambers.
Cells such as those of mammarj^ carcinomata may be completely insensitive to the
action of antibody, and in any case it is essential to ensure an adequate level of
antibody mthm the chamber, antibody which has not been produced there bv
oTel S o 1 Wakefield (1958) having ensured an adequate antibody

the
may
conforms

344

PATRICK O’gORMAN

invasion. In

and it is only in such grafts that cjdolysis is observed before this
bv t bn°^ sarcomata little or no cytolysis occurs before the invasion of the graft
by tho pmcjdnim of host Instiooytes. The function of the circulating antiWv
mnj not be tlie destruction of the graft, but rather the killing of the graft cells to

!mn n "'^»ch have yet to come, and the differences in

the hoinograft reactions elicited by different tumours (or tissues) may be a reflec-
tion of their differing susceptibility to the toxic action of antibody.

Certainly, most marked differences were found in the antibody susceptibilities
01 tiie various tumours n-hich were used in the present ivork. The sarcomata do
not show the uniform susceptibility of the leukoses, and Sal appears to be com-
pletely insensitive to the toxic action of antibodj' tn vitro, although it does bare
an effect in vivo. J3PS is susceptible to the toxic action of antibody in vivo as
has been slionni bj'' the neutralization technique (Gorer, 1956) and by passive
immunity studies, but only partly so in vitro.

Snell (1957) has pi'opounded a classification of transplantable tumours into
tliree tjqies, based on their susceptibilit}’’ to the action of antibody. The first
class comprises tumours which are liighly susceptible and includes Gorer's A
strain leukaemia, the BAGG rat tymphosarcoma, the Brown-Pearce carcinoim
(Kidd’s subline) EL4 and other tumours, all of lymphoid origin. Not onlj’ ELI
but also the other leukoses used in the present work have been shorni by Gorer to
be susceptible to the action of antibody by passive immum'ty studies, and they
are all susceptible to the cydotoxic effect in vitro. Snell’s second category' com-
prises tumours with low but demonstrable susceptibility to humoral antibodies,
and includes Sal and tho lymiphosarcoma 6C3HED. These tumours respond to
the action of antibody' by' enhanced rather than inliibited growth when passive
transfer of antiserum is used ; 6C3HED has been found to be slightly' and hregu-
larly' susceptible to the toxic effect of antibody by the neutralization teolmique
(Mitchison, 1955). The in vitro results confirm Snell’s (1957) separation of ELI
and Sal into different categories, but it is difficult to allocate BPS to one or the
other, for pre-treatment of the host v^th antiserum produces passive immunity
or enhancement of the tumour depending on the dose used (Gorer andKaliss, 195 h
and about 50 per cent of the cells are lulled in vitro. The third category' inclti es
tumours which are completely' insensitive to the action of antibody',

(1957) puts into this class the adenocarcinoma D1905, fibrosarcoma So- an
perhaps the adenocarcinoma C3HBA, which was tested by Algire, *1;,

Piehn (1954) in a diffusion chamber, and the tumour B3 with which AIil er { a
failed to show' a cytotoxic effect of iso-antibody in tissue culture.

Kaliss (1958) believes that immunological enliancement is due to a ai e
stimulant effect of antibody upon the tumour cells, so that „

in some w'ay and is thus better able to withstand and overcome t le los s ‘

The present author (O’Gorman and Alikulska, I960) by rr ^ cells

has demonstrated marked deficiencies of the H-2 antigens E an to

of Sal, a tumom: wdiich is very readily enhanced. Sal may ® Tirecluik
cytotoxic antibody because of its antigenic deficiencies, but this "^ed not p
a stimulant effect of the antibody on the cell. Similar antigenic os . ,

studied with other tumours, but it is known to occur m the early transp

generations of transplantable tumours. tvIimi oassivdy

Gorer and his co-workers have shown that circnlating antibody
transferred wall protect the host against a graft of at least some t

CYTOTOXIC ACTmTY OF MURINE ISO-ANTISEUA

neutralization technique has been ividely used to demons rate the
antibody upon cells n'liicli were subsequently graftct . llm worlc w Inch «

reported above has demonstrated that these antibodies arc toxic t» v,(ro i n

cases the antigenic system involved has been the H-2 system except, ; m r
and Amos’ antigen X. It would now seem to be beyond doubt that cirniil.it iiig
antibody plays a very significant role in the rejection of at least, certain classes ot
horaograft, notably the leukoses and some sarcomata, and altliongli its ro o in
the rejection of grafts of some normal tissues, for exam]ilc skin, is admit tedly a
minor one, it can no longer be held that the mcclianisin of Iioiiingraft, rejection
is entirely cellular, or that this major antibody has no function. 'I’lie cellular
factors responsible for horaograft rejection may in fact be cell-bound antibodies.

SUMMARY

A method has been described for demonstrating the cytotoxic action of iniiritie
iso-antisera in vitro. These cjdotoxic antibodie.s arc directed against, the M-2
system of antigens and two prernously unknomi alleles and K*’ have been
identified. The effect of iso-antibod 5 ’' on normal leucocytes and several tinnoiir.s
has been described, and the significance of these results and their bearing on the
role of circulating antibody in the homograft reaction discussed.

REFERENCES

Algire, G. H., Weaver, J. M. axd Pkehx, R. T.— (1954) J. ml. Comer . Iv sI.. 15 ItCJ.

(1957) Ann. N.Y. Acad. Sci., 64, 1009.

Amos, D. B.— (1955) Brit. J. Cancer, 9, 216.

Idem AXD Day, E. D.— (1957) Ann. N.Y. Acad. Sci., 64, 851
Idem AND Wakefield, .J. D.— (1958) nal. Cancer Inst., 21, 057.

Bro^vn, <3. C. — (1943) J. Immunol., 46, 319.

Gorer,^P. a.— ( 1956) Adianc. Cancer Bes., 4, 149.— (1958) Ann. N.Y. Acad. Sci., 73,

Idem and'A-MOS, D. B.-(19o6) Cancer Be-s., 16 338
Idem AND Kauss, N.— (1959) Ibid., 19, 824 ’

*« »™ O’OOBUS, P._(195li) Bai s ui ”■ •

Kauss. N.~(1958) Cancer Bes., 18, 992. ’

80. 419.

J R, I). G. . I. nat. Cancer Inst., IB 147,3

.bTCinsoN, N. A.— (1955) Trans. Bull., 2 93 ’

mh, u. ( 19 ,.,,) Cancer Res., 17, 2.

ACID COMPOSITION AND BILE VOLUME IN BTITTPR
^03LL0W FED RATS IN EELATIOxN TO Lim ^

S. aURWSH AND J. GILLMAR

From the Dcpartmcjit oJPhijsMogyandJomt C.SJ.R.jUniversityNntritwnBesearch UrI
University of the If itieatersrand, Johannesburg, South Africa

Received for publication April 4, 1960

^Prijiari carcinoma of the liver is a common disease amongst Africans (Berman,
IfldS), but the actiologj’’ and meciianisra still remain to be disclosed. Butter yellon-
(4-dimethyIaminoazoben7,ene) produces liver cancer mhen fed to rats, and it iras
liojicd that a better understanding of the action of butter yellow (BY) in rats
would lielp to elucidate the mechanism of liver cancer in man. BY^ does not usimllv
produce cancer in organs other than the liver, which suggests that at some stagr
in the chain of events initiated by BY a modification occurs in a metabolic path-
way which is confined to the liver, and that the modification culminates in carci-
noma. It seemed that one such pathway miglit be the degradation of cholesterol
to bile acids, which is probably confined to the liver (Harold, Felts and Chaikoff,
1955),

The cholesterol-bile acid pathwa3>- receives added importance in view of the
conversion of bile acids and cholesterol into carcinogens. The carcinogen metliyl-
cholanthrene can be synthesized chemicaU}'- from deoxymholic or cholic acids
(reviewed by Greenstein, 1954, p. 49), and cholesterol can be oxidized to several
carcinogenic compounds (Fieser, Greene, Bischoff, Lopez and Rupp, 1955). Even
deoxycholic acid itself Avhen injected under the sldn of mice can lead to sarcoma'
(Cook, Kennaway’^ and Keimaway, 1940). A particular disturbance in bile acid
metabolism therefore might conceivably'" lead to the endogenorrs production of a
carcinogen, a possibility'" that has often been suspected.

A further indication for a link between cholesterol metabolism and iicpa o-
carcinogenesis was supplied by Gillman, Gilbert and Spence (1954). These
emphasized the frequent occurrence of bile duct hyperplasia in the livers o
fed rats, and they suggested an association between bile duct hyperplasia am >
raised serum cholesterol, both of which were shoun by these investigatois o ocen
together after ligation of the bile duct, in hypothyroidism and (possibly) m a^
aminosis A. Since then Spain and GrifiSn (1957) have found an increase scr
cholesterol after feeding the hepatocarcinogen 3'-methyd-4-dimethy amm •
benzene for three rveeks, at which stage bile duct hyiperplasia also dec
This rise in serum cholesterol might be linked to a fall in the rate o ? ,n.

degradation, wliich proceeds almost entirely'" by the pathway' to bile acic s

stein, Jay'ko, Chaikoff and Dauben, 1952). mpfabolina

In this paper it is accordingly proposed to examine the bile aci ■
of BY'fed rats and controls, as expressed by the amount and
bile acids obtained after cannulation of the main bile duct. Details o ' ‘ ‘ • jjjj,
method are supplied. Attention "will be draivn to the effects of cannu ‘ jijp

liver, and an assessment will be made of the influence on bile conipo

the bile ex butter yellow teb rats

:»r

,i,er damage induced by

ce latter oteriatto ^ill l.c <li»nrr,,l i„ Irm,, of .1,0

biosynthesis of bile acids from cholesterol.

SIATEBTAL AKD METHOBS
'’'“s;l:L.leratsottl,eG.G.str.,in(Oitl.er^^

were reared on a full basal diet (*''1 “ J'" ,, 1,!,,. n,1 s are

r“ Vrfl“t™rrar'TO rat?'i This regrmc, iumluees liver Innuiurs in
flTs'ttwir S r,:i At thc Lne flL cnnlrol ra.s of the san...

age and sex ^vere reared on the basal diet alone.

ColkcHon of bih

When BY had been fed for periods vatydng from 2 to o months, (he bile dnct.s
of groups of 3-5 BY rats rveighing 200-300 g. were fistularized and (be bile was
collected in glass saddles (van Zyl, 1957). The saddles allow the rats to move
around freely in contrast to the restraining cages usually cmi)loyod. \\ ith each
group of BY rats a corresponding number of control rats was also listularized.

After the operation the majority of the rats secreted bile cojnously for several
days at least. They were maintained on the same diet except that (nSo i)er cent
NaCl was supplied instead of water. A preparation containing 1 0,0{)() i.u. jicnicillin
and 50 mg. streptomycin was injected subcutaneously after o])eration and on the.
subsequent two days.

Preliminary experiments showed that the presence of thymol did not interfere
with the bile acid analj'ses. Accordingly 0-2 ml. of 5 per cent thymol in ethanol
was added every evening as presenmtive to each saddle. Tlie bile was collected
from the saddles tvice daily and where necessary was stored at —15° C. For
mo.st of the experiments the bile from each rat was pooled from the second to the
fourth days after cannulation. Where the bile from all tlrrce days was not available
due to spillage, blockage of the cannula or other accidents, the bile was ])Oolcd
from as many of these days as possible. Bile secreted on the day before death was
di.scarded. Some rats died after a varying period of bile drainage and some were
killed with ether, as detailed later. After death the rats were examined and the
livers were preserved in formalin. Sections for histological examination were taken
trom at least tlie left lateral, left median and right median lobes.

Analysis of the bile acids
to^-VurlrS'K-Jo tile adS"

(final concentration 1-5 vl in TLITT was heated with NaOH

f thTf • itth plol

348

S. JIIR^rrSH AKD J. GILL5UA*

20 ml.). The ether extract was shaken with water (o x 5 ml 1 te mmn
sodium suiphate, evaporated and stored over P„a in a
sicator. Storap for several months did not affect the subsequent analVs^s

trihjTdroxjmliolamc acids (THA) and dilijdroxycholamc aeick (i)Hf) in
tiie samples were anatysed bj- quantitative, reversed phase, partition chroraato-
^aphj^ on columns of non-wettmg kieselgulw. The method employed was an
the qualitative method of Bergstrom and Sjovall (IDol/andSjovall
10 - 0 ^ briefly described in previous publications piirrish, 1957

1 Job). A 1 ml. siphon, a mixing vessel and 0-01 x methanolic mOH in a burette
avere used to titrate each 1 ml. fraction as soon as it was eluted. Each titration
was carried out to an end-point of pH 8-8 using a Beckman Model GpH meter, as
recommended by Dr. A. Antonis (private commimication). Die meter was con-
nected bjr shielded vire to a glass electrode and a calomel half-cell in which was
inserted a bridge of agar-saturated KCl. The glass electrode and agar hridre
dipped into the mixing vessel tlirough side arms sealed with rubber tubing. The
nitrogen was passed tlirough soda lime before entering the mixing vessel.

The solvents for chromatograpy were freshly mixed each daj- and a trace of
HGI (0-0002 x) was added to the moving phase to prevent losses on the column.
The acids were apphed as a solution in moving phase (o ml.) immediately after
50 ml. of moving phase had been run through the column. The flow rate was
adjusted to 1 ml. per 90-100 seconds. A “ base line ” figure of O-OIo-O-OSO mi.,
which corresponded to the added HCl and was best determined after elution of all
the bile acids, was subtracted from the results of each titration. Controls were run
at frequent intervals with mixtures of pure cholic and deoxjmliolic acids, by simple
cliromatography’’ and by cliromatography following allcah treatment and extraction.

Tn-o of tlie tliree ahquots from each bile sample were chromatographed and.
if the results differed by more than 2-0 mg. for THA (total 10-20 mg.) and 0-5 mg.
for DHA (total 0-4 mg.), then the third ahquot was also chromatographed. The
results for THA and DHA were expressed as concentrations or as mg./24 hours,
100 g. body weight, as calculated from the mean titration figure, the bile volume
and the mean weight of the rat on the days during wliich the bile was secretwl.
The bile volume was expressed similarly as ml./24 hours/100 g. body ueiglit.

Accuracy of the analytical method

Mxtures of 10 mg. cholic acid and 2 mg. deoxj-cholic acid were eluted from
the columns w-ith recoveries of 95 Jt 3 per cent and 119 i 10 per cent 'I ■
(A ± B signifies mean and standard deviation tliroughout this paper),
similar amounts of the acids were added to bile samples before a -a rea
the acids w-ere recovered in yields of 80-110 per cent (illimsh, vo ). > ..j.

The acciu-acy of the complete extraction and estimation vas es e ‘
of the difference between the dupheate values obtained in the presen
The standard deviation (aS) was calculated for the e^or o ejiv < „
determinations on the same sample using the formula o'- = - if,

.rp and aip are the two values and n is the number . anj p-iT m".

1-0 mg. for THA (control and BY rats), 0-15 mg. for DHA

for Dm. (BY rats). These values should be compared ^^^h the

for all results (11-6, 1-39 and 1-20 mg. respectively— see iabJe i) <

same units, namely ing./24 hours/100 g. body weight.

the bile in butteu yellow ev:o bats

results

Time of Survival and Loss of Wcirih after

Si/control ots died ,vW„ .1,0 dro. oIk d,,,. f'™, i;','.
doys 7-9, .ndtlioreommiiig -24 oonltols ««o ^ .j , 'nY

on days 16-20. In ^addition, seven BY rats were killed with elln-r «n ;

It is clear from these results that feeding BY .shnrlens the time of siirvnal ..fi- .

cannulation of the bile duct. i i ,i;,i ii,,.

The BY rats lost more weight between cannulation and deatli than did Ue
controls. Bor example, the four BY rats which survived the operation for In day.,
or more each lost 92-121 g. after cannulation, whereas no control rat lo.d more t .inn
60 g. The BY rats lost rveight steadily at a rate of -t-S g.i'day throin-hont the
period of bile drainage ; the controls lost weight at. a .‘.iinilnr r.ite until the four'
day, but thereafter the rate fell to l-5-3’0 g./day.

Changes in Bile Composition Induced Inj Butter Yellow
In order to separate effects on bile acid secretion from effects on bile volume,
the results are mostly expressed in terms of the secretion iier 24 hours jter inn p..
body weight, rather than as concentrations.

Daily variations in bile composition

Analyses were carried out on the bile samples secreted each 24 lioiirs by six
control and eight BY rats (Big. 1). The THA, DHA and bile volume fell in hoth
groups of rats on the second day after cannulation, rose to a maximum on the
fourth to sixth day and thereafter decreased at varjdng rates. The value.; for
THA and bile volume were similar in BY and control groups. On the first day
after cannulation the proportion of DHA to THA (Big. U) in the BY group iva’.s
as high as that in the control group, but thereafter the BY figure remained con-
sistently below that for the controls, suggesting that diffcrencc.s in DHA might,
exist between the two groups of rats after the first day.

The correlation coefficient for the relation between THA and DHA in the
daily samples was calculated separately for each rat. In the control group t.wo
rats showed no sign^cant correlation, three a 90 per cent and one a 9!) tier cent
correlatmn. In the BY group two rats showed no correlation, one a 0 a i
one a 98, hvo a 99 ,ad one a 99-9 per cent correlation. The iomirt „ U Te , i™

Bile composition of samples pooled over several days

secreted by each r’S duri^g^ Biibsequent experiments the bile
by and sin oentrol rats rvher. the .can SL™

350

S. MIRVISH AND J, GILLMAN

was calculated from the daily analyses
bile volumes are summarized in Table I.
IS shown for each rat in Fig. 2 and 3

The values obtained for THA, DJLA ad
ihe relationship betireen THA andDHA
For reasons ■ndiich Avill become apparent

DAYS AFTER OPERATION DAYS AFTER OPERATION

Control series

Butleryellow (BYt series

Fig. 1. — Daily variations in bile composition. Trihydrox 3 'acids (THA), dihydroxj'acids (DH.A)
and bile volume are expressed in mp. or ml./24 hours/100 g. body weight. The ciin'es reprewn
average figures for six control and for eight butter yellow rats.

belong the results in Table I are shown not only for all BY and all coiifro w -
but also separately for those rats of each group Avith a THA exceeding 1 n’c- ‘ ‘ •
100 g. and for those Atdth less than 10 mg. THA.

Tliree main conclusions can be draAra from Table I. t.-isno

Firstly, though there was a Aidde scatter of THA from rat to rat,
significant difference in THA distribution betAA'een BY and control ra s.

,HEBILKIX«mKKVKU.O^VranUTS

Tilt' —

® Bile duct hyperplasia and liver necrosis
• Other rots

Fig. 2. — ^Relationship between trihydroxyacids (THA (nnd diliydroxynrids (1)1I.\) in the
control series, for bile pooled on the second to fourth days after cannulation. THA and
DHA are expressed in rag.|24 hours/lOO g. body weight.' AA = regression line (DH.V
0-170 X THA — 0-65). The nine points below BB constitute the “ low DHA group ".
Roints for rats with any degree of bile duct hyperplasia and/or necrosis nro diffen'ntint'al
from points for rats with relatively normal livers.

Tablk l.-Trihydroxijacids (THA), Dihydroxyacids (DHA) and Bik Volume in
Control RaU and Rats Fed Butter Yellow, for Bik Pooled on the Second to
r ourth Days After Cannulation

Total

Xumber of rats .
THA (mg.)

DHA (mg.) .

Hilo volume (ml.)

Control

U-6±4-75

l-39i0-99

5-2il-40

12-4±4-04

l-20±0-61

6-20±1-58

THA less than 10 rag.

Control

7-2±1-65

0-58±0-38

4-54-1-02

7-4il-03

0-57±0-47

5-1±1-45

THA more than 10 rag.

Control

14 ' I

l'OG±0-90

5'7il-40

14-r)£2'.08
l'47-J;0-44
0-74- 1.37

352

S. MIRVISH AND J. GILLMAN

tlie variability of DHA is definitely reduced in the BY rats (98 per cent level).
Hov'ever, if on]}' results from rats secreting more than 10 mg. THA are considered,
then the mean value for DHA is significantly lower (95 per cent level) in the BY
group. No BY" rat secreted more than 2-2 mg. DHA, though the DHA of seven of
the 39 controls exceeded this value. BY thus caused a reduction in DHA, but
only when a comparatively large amount of THA was produced.

Third!}', the daily volume of bile Avas greater in the BY rats than in the normals
vdien all rats v'ere considered irrespecth'e of the value for THA (99 per cent level

0 Bile duct hyperplaiio
® Liver necrosis

Bile duct hyperplosio and liver necrosis

relative to the lines, ««. ^laSMS + grading) and/or severe liver necros h .
for rats with severe bUe duct

grading) are distinguished from pomts for rats wmi m

more than 10 mg. THA, and to an mcrease m ^de of pH.

Sso produce a "decrease

divided by daily volume). No rats secreting more ^3

is noted if all results are “hi concentration of DHA is 38 1

THA/day/100 g. are taken into accomt, mg./lOO ml. m the B c

ig./lOO ml. bfie in the controls and 22«o ± 1 94 mg /

THE BILE IN lU'TTEB YELLOW BED BATS

(significant^ different at the 90 ])cr cent level) : the eorresponiiiii!: rnnreiitr.iiii.n.,
of THA are 276 + 90 and 223 i 9S ing./lOO ml. (nn signific.ant dificrviir*-).

Both BY and control group.s showed signifirant correlations Ix-t ween *rH.\ aiul
DHA (99-9 per cent level) and between THA and volume (99 per rejil b-vc!). In
addition, the BY group showed a striking correlation (99-9 per cent level) b •t ween
DHA and bile volume, though the control group here showed no eorrelat ion at all
The first order regression linc.s were calculated for DHA ns n rnnetion „( ‘PH \

in ^^'8- 2 and 3) and for DHA as a fnneti.m ..f v.-binie

m the BY group. The regression line.s foi the DHA THA diaunins ,io ,.v ,
through the ongm, so that little or no DHA D ,.roduee<l when THA fall., behe.,

iASa ' " ''8»re. the proportion of DH \

a especially in the control series. Tlie el.ntro] r.tt . w i b

(1) DHA (expressed eithe^aTSv^orT- f-H-"-

in those cases where the dailj secretion o7tH ''-er-.e -d

^veight. This decrease was assochterw^I. L'- I'^dv

variability of DHA. (2) Bile volZo i I "> ‘l>e int.-r-individnal

con-elation developed ^twin raV a i'Xerdndividn.a!

different rats), and (4Uhe imr. - i- "'f' !'""’ed sample, fn.m

ha m,o ,„,e -'-n i >,i a

Factors Ajfechng the Inter-individual Variations in Pile ('
AnmvestieationoffLnf '-oinpnsthon

variations in bile enrit hyperplasia in both series of r-o A [ degrees of
damage. In the ^ might be explained in tor ^ '*•' <

ESt”*f tlTnT in II, e co

normal, and la rU examined from tn ^ntrol rats

“tent of e,eh fo4“'5

fyee ofbile duet hroemlTr graded 0 /+ T lY "“t®”- Tlio

354

S. MIRVISH AND J. GILLMAN

5- 13 days. Tims in at least some instances bile duct hyperplasia was present at
the time when the bile Avas being collected for analysis, i.e. on the second to fourth
day after cannulation, and any association found between bile composition and
this form of liver damage is probably direct.

Two of the control rats with necrosis had died on the fifth day after cannulation,
three had died or had been Idlled on daj^s 7-8 and three had been killed on days
11-13. It is thus not clear wdiether the necrosis developed during the period wlien
the bile was being collected for analysis or later, and the significance of any
relationship found between necrosis and bile composition wall therefore be obscure.

Nine of the 39 control rats show'ed points on the DHA/THA graph below line
BB (Fig. 2) and were hence deemed to have secreted an unusually low DHA
relative to THA. This low DHA group contained seven of the nine rats with bile
duct hyperplasia (Fig. 2) ; the liver of one of the remaining meinbers of the low
DHA group was normal and the other liver was lost. The mean bile volume

6- 5G ml. /day/100 g. body weight for the nine rats

pared v’ith 5-20 ml. for all the controls and 6-20 ml. for the B1 rats.
Inmerplasia due to cannulation of the bile duct in normal rats ^ ^

bile composition in the same v'ay as does BY administration name y j
DHA relative to THA and increasing the bile “J ° is

composition are, therefore, not specific to the process of B\ carcino^ene is
For he 16 control rats which did not show bile
secreted more than 10 mg. THA. the DHA ,vas 2-35 i
compared with 1-96 di 0-90 mg. for all control ra ® (Tatlel}.

and 1-47 4- 0-44 mg. for the BY rats vnth more than 10 mg. IHA /
Sns exetasion of the control rate rrith dn^it ^erpto.^ccc«^^^
difference in mean DHA between control and gr

significant at the 99-9 per cent level instead f of their livers,

all control rats were considered, iirespective of f j “7?‘J5)een reduced and
Furthermore, the variability in DHA of the control group has been

is now close to that of the BY group. pnntrol rats to bile duct Inper-

Cannulation of the bile duct thus led "

plasia. The latter was associated vntli a bfie ^°^P J ^ specific. If t be

by rats. These changes m are excluded, then the

SSnJ'eTn "BY^nd conSlTro.ps is greally

B. BeMionshipa bekmen bile ampmiliem, liver damage and teacitort I

in the BY rats into

Two factors, wliich do not arise in ^^5tion to cholangiofiljro

account tvhen investigating the BY « cfhS.st and neemsis of d« '*
and cancer, BY by itself produces

which elosely resemble the similar reactions resul g to co.

wnth regard to bile composition It was only^^^^^

sider the correlation between hi ’the controls, also has to 1

between DHA and volume, which is absent m

these comphcations. a ays^m ™s ado^^^^^^

of e^ch BY rat was graded with respect to each ot

be taken

the bile IK BUTTEU YEl.LOW FED BATS

DHA to THA, (2) the relation of DHA to bile vohune. (U) the extent of thh

was asiielsimilarly from a graph of DHA ploljea against ”

gradings were of opposite sign for four rats : three <if iu-se pra.liire<l le-. h. i
Is mg THA and the fourth produced 10-0 mg. THA. For eight rat. one ..r l.o h
of the points lax^ verj- close to the regression line (i.e. with gradiin:
remaining 22 rats the two grading.s were both delinitely imsit ivi' iw hot h deiinit<d\
negative. Thus DHA tended either to he high relative to both I H.\ and lute
volume, or to be low relative to both the.se factor... except where tlie total pr.«lur.
tion of bile acids avas very low.

In order to avoid dealing with gradings for both relationships (1 )H,\ 1 H A and
DHA/Volume), the two gradings were added to give a roinposite ” bile gnidiin: .
except in the four cases with gradings of ojiposite sign, where no " bile gr.idiin:
was assigned. Tliis composite grading will be n.^ed in the ensuing di'-eiis'-ion n*. n
general index of the extent to whicli bile composition dejiarts from the norma!.

A high bile grading (up to 4-j-) indicates a relatively normal Idle compo.ilion and
alowhUe grading (down to 4~) indicates a relatively ahnormnl Idle coinpo-ition.

Necrosis was present in 27 of the 3.1 livers cxamineil and Idle hyiKTpla.ia in
22. Gradings from 0 to 3+ were assigned for each tyjie of liver chimage. as uitli
the control rats. Five livers showed cholangiofihrosi.s and /or small caneiToii. fori.

IVe may now attempt to determine whether variations in tlie bile eomjxi.iiion
of BY rats are associated with variations in the reaction of the liver to can-
nulation, as in the case of the control rats. In order to facilitate analysis, (he ihita
were grouped according to the values for bile grading, diiv.s lived after eaimid-
ation and length of time for xvhich BY was fed (Tables 11, 111 and IV) The
seven cases with less than 8-.5 mg. THA, together with the rat. which ..<>en-ted
nnlln®' • gradings for DHA/THA and for l)HA/\-olume of

omitSlS a separate “low bile acid group” in Table'll and are

omitted trom consideration in Tables III and IV.

fibrosis varieTSSf” cancerous nodules and/or eholangio-

affpol Kilo ^ neither cancer nor cholangiofihrosis aniiears to

affect bile composition, apart from the general effects of BY ' ’

one exce'Jon) and a viiSleT acid group showed definite liver necrosis (with
i ) and a vamble degree of bile duct hyperplasia (Table 11). AI! Imt

t»„m of U „ .»“«»» '1 w , v ,

3-10 4+ . . 2+ 1+ + 0 TrTr~it I

Bile grading
S-r to 4 +

~ to 2 +
l-to4-
*I.o\v bile acid

All values .

' One liver was lost here.

356

S. JIIBVISH AND J. GILLMAN

one of these rats died within six days of the cannulation. Total or partial obstruc-
tion of the bile flow leads within 48 hours to liver necrosis and bile duct hyper-
plasia (Cameron and Oakley, 1932 ; Gillman, Gilbert and Spence, 1954), so that
biliary obstruction yvas probably the main cause in this group of the low production
of bile acids, the necrosis and the earty death. These cases are not considered in
the following trvo paragraphs.

Nineteen rats were not deliberately killed and did not belong to the low bile
acid group. Of these, all ten rats living for seven or more days after cannidation
shoyved positive bile gradings, including seven out of the nine rats with bile grad-
ings of 3+ or 4-t- (Table III). The five rats vdth negative bile gradings lived for

Table III.— jBjVc Gradings and Extent of Liver Damage in the Butter TeJloK Rnh.

nt Dpnth and Davs Lived After Cannulation

Time of

Bilo grading

survival

Num-

after

hor

3-t-

± 1-

JInnner

operation

to to

of dentil

(daj-s)

rats

44 -

2-1- 2 —

Died

3-6

2 5

Died

7-20

3 —

Killed with

5-0

—

2 1

3-
to

Necrosis

2-f 1+ ±

1 4 2

Bile duct
hj'perplasia

3+ 2-f 1-r i <'
I _ 4 - I

5 12 11

1 2-22

other

less than six days. Cannulation thus precipitates ^

with the most positive bile gradings, i.e. m those rats vnth bile conp

which approach closest to the normal. gradings, i.e-

The maximum liver damage was found mpts the exactly

vdth relatively high values for DHA (Table rats thus showed a

opposite position found for the control rats )■ composition,

paradoxical association of liver dainage vnth a relati Y » j j , gradings
The explanation of the paradox is c early

survived the longest after cannulation (preceing p » P ^ t hypei'P''''''
likely to develop liver injurjL Thus 2+ cannulation,

were found in seven of the ^^^re kiUed before the seventh p

but in only three of the 16 rats wdiich died ^ter than that e.xpected m

(Table III). The extent of Inmr injury was muc ^ observed m the

non-oannulated BY rats, and it is linage ^ “"'.T' *

BY rats developed as a response to ,„rriting for a considenW

Bver damage wL far less severe, even ,n i“f “,'nSuity »f

period afte? ea^ulation. BY pf My -1»““'

F4°Sy ral S -on^r .to BY m.s beteve. »

A correlation has%learly not been jj, to

composition and the condition of davs after cannulation. B

tended le in--

the bile IK BLTTEB VELU.«

Groupd According to the Lcngtii oj m j

Bile pradinp

Days oa
butter
yellorr

ei- 81

98-118

138-153

IJumber

rats

3 +
to

4 +
2

1- 3-
to to

2— 4 —

3 —

I 1
1 3

NiaT<'>-i'‘

/-"

2 - l i- .i

, 3

1 J 3

2 4 I

Hlj'' do t

u '

■ 2 • 1

1 1

«te„t after 63-Sl .a after t3S-ira ,1..« -f UV f-li.e, l.-'-Hv

place ATitbin the first 60 days. n\ {-..mK witli n bitilt DliA r.'i.itito

From the foregoing it tvould appear that . 0 ) t a. t > i n

tn TTf A also show a hieb DHA relative to bile Yolnme. (-(‘ ‘ ‘'I'l'

geneBtl egecte of BY. (3) ElgU rats altotv a low aerrel.o,, f al ' ;

associated rvitb extensive necrosis and early

lation leads to death more rapidly in those BA rats hsu-ing le < » • •

bile compositions. (5) BY rats arc vulnerable to the effec o n t t rot i, .

usuaUy develop extensive liver damage about .seven dny.s nfter eanmtlatnm. ( .)
Bile composition is probabh’’ affected nitliin fiO days of U i uHMiin)',

DISCUSSIOX

The findings will now be discussed with special refcrtdit’e to lht> cfTcct*. »if
cannulating the bile duct, cholesterol metabolism, and condilion.s other than UA
treatment which are known to affect bile acid mctaboli.sm. A meehanisin involving
changes in the rate of 12-hydroxylation will be proposed to explain observ<-d
changes in DHA/THA ratios, and the possible significance will be examined of
hydroxylation in general for the understanding of cancer.

Death follows cannulation of the bile duct more rapidly in BY rats iban in
the controls, and more rapidly in BY rats with low bile griulings than in BY rats
with high bile gradings. The rats could have died from a deficiency of a fat-
soluble vitamin, e.g. vitamin K (some rats bled very easily after a few days of
bile drainage and a deficiency of vitamin K is well luiown to result after the bih'
duct has been fistularized in man and in the dog).

The daily production of bile acids in the intact rat is only about 2 mg./HKt g.
mo (Dinstedt and Samuelsson, 1959) as compared with the 10-20 ing./

(E “'^'nd Jor cannulated rats in the present and previous invc.stigatimiH
tbrnurn production is greatly increased hy camAulation of

one auct In a rat with an intact entero-hepatic circulation the hilo acitls

hvTbcTv^’^ probably inhibit the further production of bile acids

1 tt Danielsson, 1958). Now the bile of BY rats secmiS

(Fic idA appears to show a normal DHA/THA ratio

35S

S. MUtVISH AND J. GILLJIAN

G'l production of bile acids in the canmilatecl nt

Jins result does not support the view that the raised serum cholesterol vhich‘1-

f ^i^ct hyperplasia (Gillman, Gilbert and Spence

1J54), IS due to a decreased degradation of cholesterol to the bile acids ^ ’

Eriksson (1957) showed that the DHA (actually chenodeoxj^cholic'acid was
measured) rose from the normal 10-20 per cent of the total bile acids to oO-CO
per cent on feeihng desiccated thyroid and fell to 5 per cent on feeding thioiiraci
The action of BT on DHA could therefore be due to a depression of tlmokl
tunction The latter is not knoira to be affected by BY, though thiouracil delays
the production of hepatomas by BY (Paschlds, Cantarow and Stasney, 1948-
Harris and Clowes, 1952).

Carey (1958), Rudman and Kendall (1957) and Osborn, Wootton, da Silva
and Sherlock (1959) have recently reported on the levels of serum bile acids in
various tj^es of jaundice. Though the results from the three groups of workers
differ considerablj’’, it is agreed that the THA /DHA ratio is almost ahvaj’s below
DO in cases of portal cirrhosis, and is above this figure in obstructive jaundice,
Carey found that six of the seven cases of obstructive jaundice due to carcinoma
(of various tjqies) shov'ed THA/DHA ratios greater than 3-8, compared with
two of the ten patients with other types of obstructive jaundice. Thus DH.A
production may he relatively depressed in human cancer, reminiscent of the
position in tlie BY rats. The cancer cases examined by Osborn el ai. showed
no depression of DHA.

If one speculate, the level of DHA relative to that of THA could serve sn
a useful indicator of liver metabolism in various diseases, especially if cirrhosis on
the one hand and bile duct hj'perplasia and liver cancer on the other hand are
found to affect the proportion of DHA in opposite directions. It is possible that
conditions only become favourable for the development of liver cancer when DH.-t
secretion is diminished. The bile acids maj themselves influence liver stnictnrc
and function on their return to this organ via the portal vein ; the bile ncii s
returning to the liver are already knomi to regulate the further symthesis of bi e
acids. Finally, it should not be forgotten that changes in bile acid composition
could also affect the digestion and absorption of lipids.

Possible mechanism for changes in the relative amounts of DHA and THA

Cholic acid (3:7; 12-trih5'^droxycholanic acid) is the main acid
fraction, apart from minor amounts of 3 ; 6 :

Matschiner, MahowaJd, Elliott, Doisy, Thayer and Doisy,
oxycholic acid (3 : 7-dihydroxycholanic acid) is the mam acid ofthe <

(Bergstrom and Sjovall, 1954). The depressed DHA in the BY rats . t
presumably, to changes in the relative rates of synthesis of c lo lo
oxycholic acids. According to Bergstrom (1959), these i tg fonn

lows ; The first step for both acids is the hyd^oxylation of cholestCT
7oc-hydroxycholesterol. In the case of chenodeoxycholic acid i ) iV-

is then converted from the P to the a configuration, the dou e <
saturated and finally the side chain is oxidized with the remov a |

atoms. The identical changes take place during the formation »
except that a 12a-hydroxy group is inserted at some stage e .j

dation. Chenodeoxycholic acid is not directly converted in o

the HILE IS WTVEH VEI.1..«V EIH. EATS

f nn V nml THA «<•''

It appears likely tlut the 'I^ihis n-nciun ..-.hnu

tFv f I ‘thnrofTHA Ij' Ij'-n

tPepresentpa^_^FoJet;on of m , . , ,

\ TH \ a*"'' ''.'tiU.'lh.!.

til*’

hirlinn nf hih’ jn’i'!*' m ' -.

\\ ilh an

sent paper uia; BY - -

fied'in three circumstances . ( ) prodnrlinn ''

TEA). (2) The proportion o/ WE ^ pnA n-

especially in the control sene.-,. In ra
increase in bile volume. „;,.,T„,«ctances to nuxlilimtioin in tin- rat- ,0

nthe effects are due m al ^erdinc BY leads to an .m-r.'h r.itin!, .d It-

12Eyckos}dation, it follows tlmt . I formed, the enr.yme-. eontrulbn'.! 12-

hydroxylation, (2) as more bile • - • . r.i... i,:i..

bile is swept out of the liver lomiic neiorc f, i,o.. nho b

unknown reasons the last factor does no ”1’ .' r i,vperthvroidi-m and

mentioned that there is an increased formation 1; „f 5...

in liver cirrhosis. Tliis increase could also le ‘ t-irrhod-.

hydrovelation, as already proposed by CaTC> ( ■ •». 1 • ^ «tiimil \H‘<l

^ It r^l be Recalled thk- on cannulation o the b. le due ti e er st n nlan.l

to produce an abnormally large amount of bile acn > '■ »• . A _

placed on the liver by cannulation has, according ^ . -i • ^ \Onrb

closed a slight defect in the capacity of the liver to In drox% a (. >
might otherwise have escaped detection.

The relation belicem cancer and hydroxylalion-s in the liver

An increased rate of hydroxylation in the liver may not be confined to the
possible acceleration of 12-hydroxylation by BY. Thus the intrajieritoneal in-
jection of benzpyrene, methylcholanthrene and other polycyclic liyflroearlions
(carcinogenic and non-carcLnogenic) leads to an increase in liver lienzpyrene
hydroxj-lase (Conney, Aliller and Aliller, 1957). Administration of the carcinogen
acetylaminofluorene leads to cancer of the urinarj' bladder if tiyplophan i.^ also
fed (Dunning, Curtis and Alaun, 1950). Isow 3-hydro.xyanthranilic acid and ‘.I-
hydrox7kynurenine are compounds which are carcinogenic to the bladder (Allmi,
Boyland, Dukes, Homing and Watson, 1957) and are normally formed from
trj-ptophan in the liver by a series of reactions including a hydroxylation, and it
seems that acetj-laminofluorene might act by accelerating the formation of tlicsc
two compounds bj- the hver.

.Another connection betw-een liver hydroxjdation and cancer is that the liver
can hydroxylate several aromatic amines in the ortho position to form very active
carcinogens, as proposed by Clayson (1953). The hydroxylations involved in the
conversions of trj-ptophan discussed above are of this type. Amines such as /?-
naphthylamine are similarly converted into carcinogenic o-hydroxx' derivatives
(Boyland, 1958). Acetylaminofluorene is converted by the liver into o-hvdroxv
derivatives which may be the active agents responsible for cancer of the liver d vie
0 tins carcinogen (Weisburger, Weisburger and ilorrix, 1957).

Cancer and hydroxylations may thus be linked in two wavs; (1) Follo^nr,
Clayson s hj-pothesis, the liver hydroxjdates amines to W carcinogenir o
2 G

360

s. Mm^asH j. GiLLarAN

h3^droxj^mines, and (2) tlie rate of certain I^^droxjdations in the hVer nm- l.o
increased during the stage of induction of cancer.

SHRUMABY

DetaiJs are described for a method of estimating the triln^droxy-acids (THA)
and dihj^droxjmcids (DHA) in rat bile, using reversed phase partition clironiafo-
graphjr. iJie metliod ivas applied to the anatysis of samples of rat bile, collected
on the second to fourth daj’-s after caimulation of the bile duct from 39 control
rats and 34 rats fed butter 3 mlIow (BY) for 63-153 da 3 ’-s.

After caimulation the BY rats died sooner and lost veight faster than the
controls. The caimulation and subsequent bile drainage gave rise in nianv rats
of both series to bile duct h 3 qierplasia and necrosis of the liver. The BY rats ivere
veiy vulnerable to the effects of bile drainage and usuall 3 r developed e.xtensive
liver damage about seven da 3 m after eannulation.

Although the secretion of THA ivas similar in both groups of rats, the BY rats
shoved a decrease in DHA for those rats vith a dail 3 ' secretion of THA exceeding
10 mg, /1 00 g. bod 3 ^ weight. The BY rats also showed an increase in bile volume,
and a strong correlation between DHA and bile xmlume which was entirety absent
in the controls. The proportion of DHA to THA rose as THA secretion was
increased, especialty in the control series. In the control series the nine rats uith
bile duct h 3 ’perplasia showed changes in bile composition characteristic of those
induced b 3 ’’ BY. Exclusion of the results from these rats greath^ accentuated the
difference in DHA between control and BY rats. In the BY series caimulation
rvas followed b 3 '’ death quickest in those rats vith the most abnormal bile
composition.

The significance of tlie depression in DHA b 3 ’’ BY feeding is discussed in terms
of other conditions which affect bile acid ratios. A mechanism involving alterations
in the rate of 12 -li 3 'drox 3 ^ 1 ation is proposed to explain the effect of BY^ feeding on
DHA, the increase in the proportion of DHA vdth a rise in THA, and the cor-
relation in the BY rats between DHA and bile volume. The possible sigmncance
is examined of hydrox 3 rlations in general for the understanding of cancer.

Tlianks are expressed to N. G. N. Matthews and J. Makunga for
technical assistance and to Miss J. van Veen for carrying out the cannn a
Mr. W. Lutz ver 3 '^ kindly carried out the statistical analysis ol le ‘
study xvas greatly facilitated by a grant made to one of us (S. i .) 3 , ‘ . p

Cancer Association of South Africa, to which body grateful acknoulectyn

made.

BBPEBENCES

Allen, M. J., Boyland, E., Dukes, C. E., Hoening, E. S. and Watson, J- G.-II!’-")

Brd. J. Cancer, 11, 212. , , , p;, poiindation

Beegstbom, S.--(1959) ‘ The Biosynthesis of Terpenes and Sterols . A
Symposium. London (J. A. Churchill & Go.) p. 185.

Idem and Danielsson, H.—(1958) Acta physiol, scand^.^, 1- , 8, Cll.

Idem AND Sjovall, J.— (1951) Acta chem. scand., 5, 126i.— (UoH
Beesian, C.— (1958) Admnc. Cancer Bes., 5, 66. ^

Booth, J. and Boyland, E. — (1957) Biochem. J., 66, iS.

Boyland, E.— (1958) Brit. med. Bidl., 14, 153.

THE BILE LV BrTTEB YELt.OW IT.n I'.AT,''

j{i

lO !%5

.1 .1

I -A

^ i:

. ' l‘-'

Cajierox, G. R. axd Oaki.ev, C. L.— (l!t;)2) J. I'liih. Ih'l.. 3!i, lw.>.

Carey, J. B— (105S) J. cUn. IntrM.. 37, l-IiU.

Clayson, D. B.— (1953) Brit. J. Cnnrrr. 7. -It-.O.

Co^-^•EY, A. H., JIlLLER. E. C. AKI) MlU.KI!, J. .A.,- -{I'.I.'G) J . Iri- / rl/

Cook, J. W., Kek^away, E. L. AND Kennawav. X. M. - ilBin) Xii;-:-,

Duk^sTkg, W. F., Citrus. M. R. an» .Maen. M. E. - (Ib.VH
Erikssok, S. — (1957) Proc. Snc. exp. Bin], A".)’.. 9-J. 57s.

Fieser, L. F.. Greexe, T. W.. Brsrunn'. F.. Iag’I ;, G avii lli i p
Amer. dim. Hot., 77. 392S.

Gilrert, G, Giluiax. J., Lor.sTAi.OT. V. axi> Lett.. \V. ; I •...Tsi t •

Gauux, J., Gilrert, C. axd SrExcE. L.-(i;i.-,j) 7 IRr. ‘ '

Greexstek J. P.--(1954) ‘ The Bioeliemi.trv ..f (-..n.-r ' •> S' ■ •

Thayfp IT' ’ I - A.. I-m.imtt. W. , n

(1958) /m;,V3M73 . 2Zh. vtl

» »/.. 23).

5Ios.<c;, E. S w£/'h'‘', '-T- 33 33.

Siockm. Biophy;. 51 " -''■'*-''3 . K. ,\M. Kim.,, ,. }■ j;

'■ “-F. 03

SiPERSTEix, JI. D., .Javko ’m ' V r- ' ’ ' • 3S. 5:!ii

'• - -3. W.

S«ni, J. D. p f.

• ,8

AND MoRKI.'i. II o •

'• ' • (l.'.i,) .Si-irr.r.-. J2S

7-e.

I /,

vAxzYL,i;::i93^™-f-c.--(in57)./.;,

WELSBLTiGER, J H

■il-i Ueisbcrger, E. K. AX

,•

362

SOJIE KEW DATA CONCERNING THE BIOLOGY OF TmiOUiiS.

The Effects of Hepabik and its Cojiposebts ox Tomoue Gboiitii

G. CSABA, CECILIA HOEVATH and Th. ACS

From lU InstiMe of Histology and Embryology of the Budapest Medical Umrmihj

Hungary

Received for publication Februarj' II, I960

study of tile mechanism of the agar-binding reaction — a neti' procedure for
the dia^osis of cancer — ^has raised certain problems and it is hoped that their
elucidation might tlirotr fresh light upon the role of polysaccharides in the biolotn’
^ neoplastic growth (Csaba and Toro, 1958 ; Csaba, Toro and ICiss, 1959ff, m%).
Heparin, contained in the serum, has been found to be an important factor in
making the reaction positive or negative. Immune substances, produced by the
organism in the proliferative stage of tumours, are in our opinion responsible for
the positivity of the reaction, while heparin — by gaining ascendancy over the
lipoproteins in the terminal phase of tumour-bearing subjects — causes the reaction
to become neagative (Fig. 1). By way of hj^othesis, it was suggested in our
publication concerning the details of the mechanism of the agar-binding reaction
(Csaba et al., 1959) that the positivity of the reaction in the incipient phase of
neoplastic grou'ths was due to the predominance of serum lipoproteins arising
from the tumours, or — in other words — ^that the demonstration of lipoproteins
and the consequent positivity’' of the reaction were due to the fact that the amomit
of heparin was reduced. By artificially inhibiting the action of heparin in in vitro
experiments we succeeded in turning the negative reaction given by the senini
of healthy individuals into a positive one. Therefore we felt justified in instituting
investigations into the role of heparinoid substances, particularly as the problem
of the correlation between lipoproteins and tumours seems to have been sufficiently
elucidated (Barclay et al., 1957 ; Greenstein, 1954 ; Heiger, 1957 ; Holsti, iOuS;
Homburger and Fishmann, 1953 ; Rapport, et al., 1958 ; Waterman, 1933).
A few reports are available in respect of the role of heparinoid substances. It uas,
for instance, obsem^ed by Panizzari and Vegeto (1958) that the activity of hepanti
in the serum diminished after the implantation of the Walker tumour. Se^ era
reports contain data concerning liigh glucuronidase activity observable in
(Greenstein, 1945; Hamer, 1953). It is claimed by Kizer and McKoy G9o.)) tnar
glucosamine, one of the most important structural components of hepariu, •
synthesized by^ homogenates of the Walker tumour from hexose 6-phosp la c ai

Our present experiments were based on the notion that the amoun o i p
noid substances contained in the senun becomes less during the pro i .
tumour cells, presumably because these substances are used up y w ^ n
tumour. Accordingly, the experiments were started to cover two ^

1. IVe wanted to ascertain the effect of heparin and its ®

growth and the survival of inoculated animals ; 2. hi vitro and tn mo . p

”

of tamouts. a,e effects pma»«-l >'>

The present paper d

nents.

Fio. 1. — Correlation between the respective systems of lipids-antilipiis ond mueopolyfinc-
charides-antimucopolysaccharides, and its effect on the result of the agar-binding reaction.
The amount of lipids increases (shade lines) or that of the heparinoids decreases (squares)
in the incipient phase of tumours.

JlKTHUn

^Ye studied the effect of heparinoid components on 220 tumour-bearing mice
Their average body weight was 20 g. The test animals-white and sand-coloured
males and females-were taken from the stock of the National Institute of Ptihlic
Health. Budt^est. Transplanting Ehrlich ascites tumour into the mice, we used
coses 0 • a ml. for subcutaneous injections, i.e. approximately 60,000 cells

364

G. CSABA, CECILIA HORVATH AND TH. ACS

per mm.3 ; the dose for intraperitoneal injections was likewise 0-05 ml Imt n
ascitic fluid was first diluted at the ratio 1 : 10 so that tlm nnmhpr
approximately 6000 per in these eases ’ Jhte
modified and are indicated in the tables. Low doses had the purpose of proToiS
conspTcuoS. differences between experimentals and controls were more

The animals were treated with the subcutaneous administration of Heparin

fr^hl) (Fluka) and a, d-glucosamine

(L gilt) preparations. The daily doses of these substances were dissolved in 1 ml.
of physiological saline. Tumour-bearing controls received a daily dose of 1 ml.
of physiological saline, injected subcutaneously. We also used controls treated
with glucuronic acid, glucosamine and heparinoid substances but without tumour
injection. Other than the lieparinoids to which the animals succumbed, the other
substances showed no effect.

RESULTS

The effect of glucuronic acid and glucosamine on the survival of tumour-
bearing animals is shoivn in Table I,

DISCUSSION

It was showm by the experiments that glucuronic acid, in daily doses var 3 ’iiig
beUveen 5 mg. and 30 mg., invariably promoted the destruction of tumour-
bearing animals in all groups, but failed to do any harm to non-tumour bearing
controls. It therefore seems justified to attribute the observed effects to the
presence of tumours as neoplastic growi/h was alw'ays more marked in trentcrl
than in non-treated animals. The effect of glucosamine appeared to be similar
to, although less conspicuous than, that of glucuronic acid. That the observed
effect must be principal^ due to glucuronic acid was strikingl}'’ demonstrated b}
the experiment in wdiich both glucuronic acid and glucosamine were administered .
their combined effect was in no way different from the effect observed in the
animals which had received glucuronic acid alone. .

That we were right in using only small amounts of ascites for tlie transfers i>
convincing^ shown by the experimental results. Although high doses of glucuronir
acid were administered to the members of group 7, the difference between expf"
mentals and controls was very slight in this group where the transplan e ,
had not been diluted. Survival of the controls was considerably longer in groiij
w^hile the survival of the experimentals was 25 per cent shorter than in group ^
Differences are most pronounced in the intraperitoneal groups 5 an w '
time of survival of the controls is very long and even reaches t la o sn >

ously inoculated animals. nl?n bv

It was found by HeiJbrunn (1956), Balazs and Holmgren (19D), ana ^
Koenig (1955) that whole heparin inhibits tumour growth. . j^r^c

this effect because the doses of heparin applied in our expenmen s
haematomas and led to a quick death of the animals. At any rate t
accept the statement of these authors we must emphasize la tiimoiiR.

apply to the structural components of heparin w'hich, far irom m t-xperi-

promote neoplastic growth and so hasten the death of the anim.

Group

nK GllOWTH

OE HEPABIE OE T

V Acid Glucominnr on (/"' I

iB'.r.

Xuiubfir of

aniinufs

f Treatment survival OU-^rvatmu.

Colons. dosesjroousc ‘lny=' ^ Mil ' ’••’■

tr* atoS^sMion do^eafmouso .1.^
p -Hnnnnn

AVhite,r‘

»5

AVhvte, F.

S.G.

Sand, M-

i»

*♦ »

JJ »

Sand, F .

S.C.

„ »»

Sand, F .

S.C.

•1 >»

AVhite, F.

I.P.

*t If

Heparin

10 mg./day

Control

Glue. ac.

10 mg./day
Ditto

Control
Glue. nc.

10 mg./day

Control
Glue. ac.

10 rog./day
Ditto

Control
Glue. nc.

5 mg./dny

35 •«

30-8

33-3

32*0

20*3

27*1

33*3

20 -f.

32*0

27*0

2!)*8

33*4

23*2

WTiite, F.

I.P. Control

5\Tiite, F. I*P*

\VlHte,F. I.P.

AVhite, F.

Glue. nc.

30 mg./dny

Control

Glue. ac.

30 mg. /day

Control
Glucos. 2 mg.
+gluc. ac. 5
rag./day

Glue. ac. 30
mg. /day for
30 days

20*0

15*0

13*3

11-0

20*0

18-0

11*5

17*8

o-n

30*0

25*0

17*3

33-4

23-8

28*8

liKK-nlaOd witlt
nndilut<<l ii> '"il.*'.

Inm'ulnle*! " illi
Ulldillll**.! Ilx-itr

Identiral uilll roti-
Irol of group 5.

Bereiviui no 111 -
mour ; no efTeel
oliserved.

mental results raise the problem : is the higher level of serum mucoi(l.s in the blood
of tumour-bearing individuals and test animals with well-advanced tumour? (o
he regarded as a consequence of neoplastic growth (Almquist and Lan.sing. Hiri? ;
Hombnrger and Kshmann, 1953; Rottimer, Let^^ and Conte, 19.58; Slietlar
r( ft/., 1949 ; Weimer et al, 1957 ; Winzler and Smyth, 1948)— from the disinte-
gration of mast cells etc.— or as the causative agent of tumours? There seems lo
he no sliarp boundar}' hetiveen cause and effect : both seem to participate in the
maintenance of the vicious circle in malignant disease.

It was not possible to resolve these problems on the evidence of the present
expenments. Therefore, additional experiments-fw vitro and in uiw-have been
performed and the results form the subject of a subsequent publication

366

G. CSABA, CECILIA HORVATH AND TH. ACS

siraEMARy

TJie effect of heparin and its components on the surnval of tumour-bearinc
animals lias been examined. The experiments, as described in the paper, show
that glucuronic acid or glucosamine and glucuronic acid — ^if administered in
protracted doses — shorten the life of tumour-bearing mice.

REFERENCES

Almquist, P. 0., AND Lansing, E.— (1957) &and. J. din. Lab. Invest., 9, 179.
Barclay, M., IfAuraiAN, R. J., Sved, D. W., Kidder, E. D., Eschek, G. C. axd
Petermann, M. L. — (1957) Cancer, 10, 1076.

BalAzs a., and Holmgren, H. — (1947) Proc. Soc. exp. Biol. N.Y., 72, 141.

CsABA, G. AND Toro, I. — (1958) Z. Krebsforsch, 62, 481.

lidem and Kiss, F. I. — (1959) Orientacion Medica, 8, 1. — (1959) Orv. Hetil., 100, 15S0.
lidem, Jocsai, G. and Elodi, B. — (1959) Neoplasma, 6, 366.

Greenstein, j. P. — (1954) Biochemistry of Cancer ’. New York (Academic Press).
Hamer, D. — (1953) Bril. J. Cancer, 12, 661.

Heiger, I. — (1957) Proc. Roy. Soc., 147, 84.

Heilbrunn, L. V. — (1956) The Dynamics of Living Protoplasma ’. New York (Am-
demic Press).

Holsti, P. — (1958) Nahirwissenschaften, 45, 394. ,

Hombiirger, F. and Fishmann, W. H. — (1953) ‘ The Physiopathologj" of Cancer .
Neiv York (Holter).

ICiZER, D. E. AND McCoy, T. A. — (1959) Cancer Re^., 19, 309.

Koenig, H. — (1955) Z. exp. Med., 126, 67. . „ ■ „

Panizzari, G. D. and Vegeto, A. — (1958) Arch. ital. Pat. chn. Tumon, 2, 1-19.
Rapport, M., Graf, L., Skipski, V. P. and Alonzo, N. F.— (1958) Nature, 181, IbUJ.
Rottimer, a.. Levy, A. L. and Conte, A. — (1958) Cancer, 11, 351.

Shetlar, M. R., Foster, J. V., Kelly, K. H., Shetlab, C. L., Bryan, H. b.

Everett, M. R. — (1949) Cancer Res., 9, 515.

Waterman, N. — (1953) B^dl. Ass. franc Cancer, 40, 340. J mt

Weimer, H. E., Quinn, F. A., Redlich-Moshin, J. and Nishihaea, J.

Cancer /list, 19, 409.

WiNZLER, R. J. AND Smyth, J. M.— (1948) J. din. Invest., 27, 617.

SOME NEW EATA COKCEEKING

The Effects of Hepahi:n iMinmoRS .

r GSABA TH ACS, CECILIA HORVaVTH ESZTICU KAl'A

... J'X . H.... .. « ^

Received for publication Fcbnwn- U. lOdO

We have reported in our preceding communication f

Acs 1960) that the components of heparin shorten tlu llfcniiire

animals by promoting neoplastic growth. Since, according o ' ,„j

Lse substaLs may be sjmthesized by the tumours
AIcCoy, 1959) and since— on the other hand— they nccumula i_
of individuals uith advanced tumours (Alrnqmst and Lansing. i Lot turn r.
Le^^^ and Conte, 1958 ; Shetlar el al, 1949 ; Weimer c( of., I'A), •. W inzlcr ami
Smyth, 1948), we thought it justified to ascertain whether and if so lum Urn
neutralization of these substances would affect the vitality of tumours. In accor-
dance udth our programme, described in the preceding comnuiiiiratioii. ue
neutralized the heparinoid substances in two kinds of experiments; in tisMic
cultures and in the living organism itself.

METHOD

In vitro experiments to test heparin inhibitors were performed in OOP tissue
cultures, half of which were Maximow’s double cover slip cultures and the other
half flat-tube cultures (Toro, 1959). Eowl plasma coagulated with chick embryo
extract, to which no heparinoid had been added, served as protective mediutn.
A mixture of horse serum, chick embryo extract and Tyrode solution (fl; 1 ; (i)
V as employed as culture medium for the controls, Toluidine blue was u.scd as
heparin inhibitor in the experimental cultures, this being added to the liquid
medium to give a concentration of 1 y/ml. or lOy/ml. (called “ final conccn-
tration ’ m the follovdng). It was either immediately or 48 hours after cxplan-
tation that the wasbng fluid containing toluidine blue was added to the ciilture.s
Me observed the cultures during 10 days or fixed them within this period 'I'lio

Si rf CsH. Guerin, Yoshida and Ehrlich tumours

uhile the control cultures were derived from the thymus liver snleen K-mnl.'

T2 'ilf on n,ioc „„,1

Mock of tlie National Institute of Public IlAtlf the ktt’^f*’ sand-colouroti

<00. and

368 G. CSABA, TH. ACS, CECILIA HORVATH AND ESZTER KATA

and, ammonium chloride (United Works of Pharmaceutical and Dietetic Products
Budapest) were applied.

At the outset, toluidine blue alone was used for the purposes of inhibition.
It was first added to the food of the animals in a pulverized form. It was not well
tolerated and the dose applied could not be estimated accurately. Therefore, we
tried to administer the dye by way of a gastric tube. The technique of administra-
tion was rather unsatisfactory and quite a number of the animals died during the
process of intubation A\dnch e.xplains why there are certain groups in the tables
with such a low number of test animals.

Having been conidnced by the results of our preliminarj"^ experiments that,
neither toluidine blue nor protamine sulphate alone avere adequate^ efficient if
given in tolerated doses, ^ve had to start combined treatments. The tested com-
binations are denoted b}’’ the eharacter “ T ”, i.e. the first letter of toluidine blue,
their chief ingredient. The combined preparations bear the numbers to Tjj,
of which only T5, T4 and — to a lesser extent — Tj^ proved to be useful tumour
inhibitors.

The preparation T^ contains 10 mg. /ml. of toluidine blue and protamine sulphate.
Rats were given 10 mg./lOO g. body weight of toluidine blue and 0-5 ml. per animal
of protamine sulphate, the dye being dissolved in 1 ml. of distilled water. Mice
received the same concentration of toluidine blue in the follovdng doses : O-S ml.
on the first, 0-6 ml. on the second, 0*4 ml. from the third day, and — in addition—
0-1 ml. per animal of protamine sulphate.

The combined preparation T4 contained 10 mg. of toluidine blue, 10 mg. of
thionine and 10 mg. of ammonium chloride per ml. of distilled water. The reason
for choosing these substances will be explained in the discussion. Rats were given
1 ml. per animal per 100 g. bod}' weight, while mice received 0-4 ml. per animal
per 20 g. body weight of this preparation. This combination contained no protamine
sulphate.

The preparation Tjj prov'ed to be highly toxic and therefore not suitable for
our experiments. It contained 20 mg. of toluidine blue, 10 mg. of tluonine an
20 mg. of ammonium chloride per ml. of distilled water. As mentioned abo^e,
we also tested a few other combinations. These either produced no effect or were
exceedinglj' toxic. The parenteral introduction of dyes, resulted in unsuccess u
experiments.

RESULTS

Toluidine blue, applied in a final concentration of lOy/ml. in ^

experiments, either immediately inhibited the grovdli of the examme ^ , j

01 did so in a very short time. The proliferation of tumour cells m „ g

cultures seemed to be especiaUj' intensive and conspicuous in the
and Elrrlich tumours. Where the fluid containing toluidine blue
the cultures immediately after the explantation, only a few spora c o
cells appeared and these contained the dye in the form of
cases where the dye w'as apphed 48 hours after the explantation, e
difference betrveen the groudh of the test and control cultures un i a
tion of the dye. Then isolated granules in the cells of the test . gjoirth

the dye and showed metachromasia (purple). It should be note m
of the cultures stopped forthwith so that — as regards groAvtli--- ^ i . thereof.
24 hours after the treatment did not differ from that seen at the eg

EFFECT OF HEPAKIK INHIBITORS ON TUMOUR GROWTH

369

A day after the application of the dye, it was still very finely distributed in some
ceils," while in others it appeared as coarse particles as were observed in cultures
that’ had been treated immediately after explantation. No further grondh could
be obserred even 48 hours after the treatment, while the controls continued to
grow vigorously. It was at this time that the cells began to disintegrate in the
treated cultures, a process which was completed 96 hours after the single treatment.

The binding of the dye in the cells is irreversible. If the culture is normally
fed 24 hours after the first (and only) treatment and if also the subsequent wash-
ing? are similar, proliferation will never occur again.

Toluidine blue in a final concentration of 1 y/mi. gave less obvious results than
the higher concentration. Only 1 or 2 granules were Seen in the cells 24 hours
after the treatment, and the tumour continued to grow, although less intensively
tiian in the control cultures. The accumulation of granules became more pro-
nounced 72 hours after the treatment ; cellular disintegration began 96 hours
after the first and 48 hoius after the second treatment, to become complete by
the end of the period of observation.

With one exception, none of the organs enumerated in the paragraph on method
took uj) toluidine blue. This exception was the thymus. Like tumour cells, the
<eik of this organ — the epithelial membrane and the thymocytes — ^took up
toluidine blue and disintegrated subsequently.

It .should be noted that inhibition of growdh by the dye was ahvays more
lironounced in the case of carcinomas than in that of sarcomas.

inhibition in in vivo experiments are tabulated (Tables I to
- i). Die first e.vperiments in connection -with Guerin’s tumour were per-
<irmed witli large doses added to the food. Seeing that this method, while being
I allow of the administration of precise doses we began adminis-

r.i mil ga.stric tube. We took care, in our further experiments, to begin

I. Inoculated loith Guerin Tumour Subcutaneously
Treatment begun 8 days after inoculation

NiiiiiU.r

<.r

Truntment
*' * T(I. Itlvit-

ill foixi

Average

Dura. weight Standard
tion tumour deviation
(tinys) (g.) (g.)

— . 20- 13 . ±2-4
13 . 12-0 . 1-5

Average
meta-
stasis Observation

3-3 . _

0-62 . Regionally only.

n, Udi), Inoculated u-itfi Guerin Tumour Subcutaneously
Trt.unipiit begun 48 hour.? after inoculation

Dura-

Average

, 4

weight

■ ' Tt,. .

tion

tiimour

* .'tinujjj

(days)

Ip.)

^ • T.!

I 1 i t

• — .

4-44

• 23 .

.‘i-1 ::

, nv. <l,iv

• T'

'I ,

• 2.3 .

0 -os.-,

■ 23 .

O-U.-I

Standard Average
deviation rr.ota-

(?•) stasis Obser\'ation

1*0 . IST mg. .

1-42 . 250 „ . Xoxic phenomenon,

0-012 . —

o-ois . — ■ ■■

G. CSABA, TH. ACS, CECILIA HORVATH AND ESZTER KAPA

Table III, Rots iTiocvlatEdf ivitli ChiBTiTt Tuthoiit STibcufct/tisously
Treatment begun 13 days after inoculation
Average

Number Dura- weight Standard

tion tumour deviation Inhibition

Sex annuals Treatment (days) (g.) . (g,) . Observation

al- • 4 . Control . — . 8-73 . 1-68 .

M. . C . Ts . 7 . 6-04 , 1-2 . 24-2 . —

Table IV . — Rats Inoculated with Guerin Tumour Subcutaneously
Treatment begun 19 days after inoculation

Number

Dura-

tion

Average

weight

tumour

Standard

deviation

Average

meta-

Sex

animals

Treatment

(days)

(g-)

is-)

stasis

Observation

. 2 .

Control

* ““

18-15 .

3-2 .

9-25 .

Abdominal cavity
filled with meta-
stasis.

31.

. 7 .

5-85 .

1-8 .

2-65 .

3Ieta8tasis region-
ally only.

Table V. — Rats Inoculated loith Guerin Tumour Subcutaneously
Treatment begun 28 days after inoculation

Sex

Niunher

animals

Treatment

Dura-

tion

(days)

Average

weight

tumour

(g->

Standard

deviation

(g-)

Average

meta-

stasis

Inhibition

(%)

3 .

Control

. — .

14-1

1-63 .

1-5 .

62-4

5 .

. 7 .

5-3

. 0-74 .

3-0 .

—

Table — Mice Inoculated with 0*05 ml. of Ehrlich Ascites Tumour Subcutaneously

Treatment begun 24 hours after inoculation

Sex

Number

animals

Treatment

Duration

(days)

. 10 .

Control

—

31.

—

10 .

Tol. blue

31.

. 20 .

25 mg. /day
-fprot. sulph,
0-1 ml. /day
Ditto

31.

. 20 .

Tol, blue

17 .

25 mg./da 5 '

Average

weight

tumour

(g.) Observations

3-8 . —

• — . , f

1-88 . Dose toxic; only 7 annuals alive at

examination.

1-46 . Dose strongly toxic ; only 3 animals

alive at examination.

1-83 . Dose strongly toxic ; only 3 animals

alive at examination.

Table VII. — 3Iice Inoculated with 0-1 ml. of Ehrlich Ascites Tumour

Subcutaneously

Treatment begun 9 days after inoculation

Number

Sex animals Treatment
51 8 • Control

m‘. '. 10 . T„

Average
Dura- weight
tion tumour
(daj’s) (g.)

, — . 3-41

. 14 . 1-87

Standard

deviation Inhibition

(g.) (%)

0-63 . —

0-41 . “IS-S .

Observation

EKFECT OF HEPAEIN I2CHIBITOP.S ON TXJMOUP. GROWTH

371

Table YTH.—Mice Inoculated with 0-2 ml. of Ehrlich Ascites Tumour

Subcutaneously

Treatment begun 12 days after inoculation

Sex

Kumher

animals

Treatment

Dura-

tion

(days)

Average

weight

tumour

(g-)

Standard

deviation

(g-)

Inhibition

(%)

Observation

. i .

Control

. —

1-7

. 0-37 .

—

. 31 .

. 7

0-91

. 0-14 .

46-5 .

' —

Table IX. — Mice Inoculated with 0-1 ml. of Ehrlich Ascites Tumour

Intraperitoneally

Treatment tiegun 24 hours after inoculation

Amount

XarniKm

Dura-

tion

ascites

Cell count
in 1 ml.

Average
cell count

aniinnh

Treatment

(dayg)

(ml.)

of ascites

per mouse

Observation

. 10 .

Control

—

3-73 .

, 99,980 .

373,10' .

—

. 10 .

Tol. blue

0-4 mg.

-f firot.

0- 1 ml./dav

8 .

2-78 .

. 92,800 .

258,10' .

Three mice died.

• 10 .

T,0-8ml.. ' .
tlion 0-4 ml.

1*5

. 114,e00 .

' *

Six mice died during
treatment ; no as-
cites in 2 mice.

Iaiile X. — Mice Inoculated with 0-1 ml. of Ehrlich Ascites Tumour

Subcutaneously

Treatment begun simultaneously u-itli inoculation

N'lltlAdT

^ nuiinnU
M. . 4

M. .

Tri’dtrnpnt.
f 'oiu rol
Tj liound to
jmlyvyiiil
liyroliilono

Averngo

Durn- weight Standard

tion

of tumour

deviation

Inhibition

(days)

(g-)

(%)

Ob-servation

— .

10 .

0-2,5 .

10 .

0-43 .

00.5 .

Two out of 5 animals
died.

Iaiua XI.— .i;,Ve Inoculnkd with Crocker S. ISO Subcutaneously
Ireatiuent begun 10 day.s after inoculation

N'u'.U'r

I-

Trt-.uiiunl

‘'■'V.Mil

.\vcrnge

Dura. weight Standard

turn of tumour devi.ation Inliihiti
(day-) (g.) (£,_)

— • 3 ft . 0-7 _

■ •

Oh^rvation

Three mice died dur-
mp proccivs of ferd-

372

G. CSABA, TH. ACS, CECILIA HORVATH AND ESZTEB KAPA

Table Xll.—Rats Inoctilated with Yoskida Tumour Subcutaneously
Treatment begun 4 days after inoculation

Sex
M. an
F.

Ditto

Number

nnimals

1 10 .

Treatment

Control

Dura-

tion

(days)

Average
weight of
tumour
(g-l

9-6

Standard

deviation

(g-)

2-7

Inhibition

(%)

10 .

. 7

5-5

1-35 .

42-7 .

. 10 .

* 7

5 04 .

1-58 .

47-0 .

Observation

Double dose on Jst
day.

Double dose of tol.
blue on 1st day.

treatment at different times, i.e. to obtain tumours tvitii different degrees of
vitality. Figures of measurements, percentage of inhibition (wherever such
calculations were deemed to be justified by the number of animals) are indicated
in the tables ; special observations are contained in a separate cohmn.

Ehrlich ascites tumours ivere transplanted partly through the subcutaneous
and partly through the intraiieritoneal route. As has been mentioned, the purpose
of low dosage was a longer survival of the animals.

Only a few groups were inoculated wth Crocker S. 180 and Yoshida tumours
because results obtained in these cases were in perfect agreement with those
obseiT'ed in coimection with the other two lands of tumours and also because the
number of animals in the particular groups seemed to suffice for adequate con-
clusions.

DISCUSSION

It was suggested in our previous publication that heparinoids may promote
neoplastic growth or that they may even be regarded as causative agents. Our
present experiments vith heparin inhibitors tlrrow a much sharper light upon
the role played by potysaccharides.

It was proved by these experiments that even extremely low concentrations
(10“®g) of toluidine blue are capable of inhibiting tumour grorvtli and destroying
tumour cells in tissue cultures. Apart from this, it was sho-wn — a fact of great
theoretical importance — ^that what happens in the cells is not a mere storage
of toluidine blue, followed by removal by phyagocytes, but the binding of heparin,
a substance which is essential for the proliferation of the tumour cells. That this
is so is borne out by the fact that the dye shows metaclu-omasia in the cells.

The balance of the experimental results allows the conclusion that heparin is
sjmthesized by and utilized for the growth of malignant tumour cells. There
are, therefore, three alternatives open for us if we want to stop tumour growth
in vivo :

1. The binding of some cytotoxic substance to glucuronic acid or hepannoicl :
its selective accumulation in the tumour cells should lead to their destruction.

2. The suppression of the organism’s heparinoid substances with the conse-

auent inhibition of tumour grovrih. . • •

^ 3 A combination of land 2, i.e. binding of the orgamsmsoumhepa^oid^

especially of the heparin which circulates in the blood, followed by the introduction

373

effect of HBPAKIX INHIBITOBS OS TTENlOim GROWTH

into the organism of heparinoids bound to a cytotoxic substance for whic^on
account of tile bound condition of the organism’s own beparmoids— the affimty

of the tumour has increased. _ , , -n i _c «■(-

Seeinff that method 1 requires chemical operations that mil be performed at
a later date, we began the examination of method 2, little suspectmg that, by
doing so, we were to arrive at method 3, which then proved the inost useful.

Kesults assembled in the tables make it clear that the bmdmg of the heparmoid
.substances strongly inhibits tumour growth. The percentage of inhibition is
usually about 45 per cent but reached in one case as much as 62-4 per cent {iable

^ ^ The potenev of the inbibitorj^ effect seems to depend on the rate of grm^h of
each particular tumour, the time between tumour inoculation and the beginning
of treatment, and on the size of the tumour at tliis time. The nature of the tumour
does not appear to have a decisive influence on the strength of the effect, for
high values of inhibition were obtained with every kind of tumour. We concluded
from our experiments that the best results could be expected if (1) treatment were
started immediately after the implantation of the tumour ; (2) slowly-gromng
tumours were treated ; (3) the treatment were protracted.

We feel justified in claiming that our method, elaborated upon the basis of
theoretical con.sidcrations, jdelds satisfactorj’’ results in the case of transplantable
I uwmirs. It seems nevertheless necessary to discuss a few problems Avbich influence
the succe.s.s of the treatment.

ft is, first of all, verj' important that adequate doses be used. We observed
tliaf tlic growth of the tumours did not begin to decrease immediately after the
treatment ■. it required 0-7 days for the animals to become saturated wdth the
dye. It Ava.s then that proliferation slowed or stopped and necrosis occurred.

Why is combined treatment more satisfactorj’’ than the other methods?
Neitlier folnidine blue (in non-toxic doses) nor protamine sulphate alone produce
iiihiliition, while their combined application never failed to give satisfaction.

I his rai.se.s tlie theoretical possibility that the essential point of the experiments
u as u\ore than the sinqde binding of heparinoid substances, namely the combination
of methods 1 and 2. Ex]ieriments performed on rabbits (unpublished) have
shown that ]irot amine sulphate is capable of strongly diminisliing the heparin
i'-Ycl of the blood for a jieriod of G-IO hours. The absorption of toluidine blue
IS slower ; tliis was administered perorally and not parenterally as the protamine
M!l))hat(>. It is. lieiice, safe to assume that, during the time of absorption, toluidine
mie does not or only to a negligible extent — combine with the heparinoids of
the blood so that the dye is much more bound in the tissues. This would mean
h it. alter some time, the heparinoid.s of the tissues— including those around the
iim(uir> heeome hotmd to toluidine blue. The binding is verj- stron^f. It is on
!'■ <i ler hand, coneeivable tliat a.s long as the heparinoid substances of the blood
. ’ "'""it \)y protamine sulphate, the tumouns are able to take up onlv those

Provided it is true that toludine
udaG d m the cells produce.sa toxic effect— and the results of experiments
,<'"l«<res eonlirm tins assumption-all that actually happens is that

u!h ti

the organisms own heparinoids to a evtotoxic agenTbv means of
> ; a! Vroe^lure so that, circulating heparinoids being hound timaffinitv

’ ' -1-1. are hound by S

mmdi.ite Mcmity becomes more pronounced.

’ in their inn

374

6. CSABA, TH. ACS, CECILIA HORVATH AND ESZTER KARA

The question arises here : why is there such a great amount of toluidine-bliie-
bound heparinoids around the tumours? The answer is simple enough The
increases in the neighbourhood of tumours (Asboe-Hansen
1954; Asboe-Hansen, Levi and Wegelius, 1957 ; Cramer and Simpson, 1944-
Csaba, Toro and Kiss, 1959 ; Koemg, 1955 ; Lascane, 1958) ; these cells contain
heparinoid substances which combine uath toluidine blue. The afBnity of the
mast cells for heparin-binding substances is very strong, perhaps stronger even
than that of tumour cells ; hence, if we require that there should remain a sufficient
amount of heparinoids for the tumour cells beyond what has been bound by the
mast cells, the doses used in the treatment must be adequate, and— equally— the
duration of the treatment itself must be long enough. Since the uptake of hepari-
noids by the mast cells weakens the tissue barrier which helps to prevent tumour
growth, doses must be such as to damage not only the mast cells but the tumour
cells as well.

It may be presumed that the reasons why the effect of dyes does not manifest
itself until the 6th or 7th day following treatment is that this time is needed for
the organism to acquire sufficient toluidine-bound heparinoid as to enable the
tumour to utilize this material.

Another form of treatment tested by us was the combined administration of
toluidine blue, thionine and ammonium chloride. We were led by the following
considerations in doing so : (1) Thionine, too, combines with heparinoids, although
not quite as specifically as toluidine blue. As it is less toxic it can be well emplo 3 '’ed
together with toluidine blue uithout increasing the toxicitj’- of the latter. (2) A
histological observation was our second reason : we found that a reduction of
the pH value during the process of staining ndth toluidine blue helped to make the
staining of mast cells more specific. Tliis induced us to use ammonium cliloride
for acidification in vivo, and the result proved to be satisfactorJ^

The objection might be raised that the tumour-reducing effect of heparin
inhibitors is due to a general intoxication of the organism produced by these
substances. We think that their specific action is well proved by the electivity
observed in the comse of experiments with tissue cultures, further bj'- the fact
that we succeeded in inhibiting tumours in living animals vdthout anj'' sign of
a general intoxication ; and — finallj'^ — by the fact that the administration of
easily tolerated doses of the two substances (protamine sulphate and toluidine
blue) proved to be more effective than either of them administered independently
in toxic doses.

Our experiments have thus furnished evidence to show that tumours utilize
or S 3 mthesize heparinoid substances required for their growth. Led by such

EXPLANATION OF PLATE

j-jg, 1 . CjH-culture 72 hours after explontation and 24 hours after normal feeding. Control.

Fi^^^^^aH-cuR^e placed in a liquid medium containing 10 y/id.

ately after explantation and fed with the same fluid after 48 houm. Th®. pho
taken 72 hours after explantation. Accumulation of metachromatic toluidine blue

able in cells. Unstained, x 75. . .. ^ j- ...in, loltiidino

Fio. 3. — CjH-culture, 72 hours after explantation and 24 hours after yuo

blue in a final concentration of 10 y/ml. Many metachromatic granules of toluidin
observ'able in cells. Unstained. X 75.

British iIouhnal or Cancmr.

Vol. XIV, No. 2.

Csaba, Acs, Horvath and Kapa,

EFFECT OF HEPABIN INHIBITORS ON TUI^IOHU GROWI’H

375

theoretical considerations we succeeded in elaborating a method for the inhibition
of tumour growth which has proved successful in animal experiments. The sub-
stances employed by us are antimetabolites rather than cytostatic substances.
That this is true is shown by the fact that cytostatic substances are Icnown to
affect quickly-dividing tumour cells in the first place, while our method produces
a more marked effect if cell proHferation is slow. Far from being inconvenient,
this feature of our method is decidedly advantageous if we remember tliat — apart
from haemoblastoses— tumours in human subjects are frequently slower growing
than transplantable animal tumours.

SU^UURY

Relying on the evidence of in vitro and hi vivo experiments the authors conclude
that tumours require heparinoid substances for their growth, and describe new
possibilities for the inliibition of tumour growth. Theoretical considerations have
led to the elaboration of a method by wliich it is possible to check the proliferation
of transplantable tumour cells in about 45 per cent of the test cases. These
theoretical considerations, substantiated by experimental results, open up a
fresh path to new therapeutic experiments.

The authors received valuable assistance in their experiments from the staff of
the United Works of Pharmaceutical Dietetic Products, Budapest, for which they
Avant to express gratitude.

REFERENCES

Almquist, P. 0. AXD Lansing, E.— (1957) Scand. J. din. Lab. Invest., 9, 179.
Asboe-Hansen, G. — (1954) ‘The mast cell’ in ‘International Review of Cytology’,
Ed. Bourne and Danieli, New York (Academic Press) Vol. 3).

Mem, Levi, H. and Weoeltus, 0.— (1957) Cancer Res., 17, 792.

Ckamer, W. and SntPSON, W. L.— (1944) Ibid., 4, 601.

CsABA, (I., Hokvath, C. and ACS, Th. — (1960) Brit. J. Cancer, 14, 362.
iaem, Toko, I. and Kiss, F. I. — (1959) Orientation Medica. 8, 1,

&ZEU, D. E. AND McCoy, T. A.— (19.59) Cancer Res., 19, 309.

Koenig, H. — (1955) Z. exp. Med., 126, 67,

Lascane, E. F.— (1958) Cancer, 11, 1110.

Rottimer, a.. Levy, A. L. and Conte, A.— (1958) Ibid., 11, 351.

SiiETDAR, lil. R., Foster, J. V., Kelly, K. H., Shetlak, C. L.

.. :Ev£RETr, M. R.— (1949) Cancer Res., 9, 515.

ORO, L--(1959) Szovettenyesztes (Tissue Cultures) In A. Kovach’s book : ‘ Methods
Suie“demy)’” Medicine ’. Budapest (Hungarian Publ. House

Nishihaea, H.~(1957) ,7. nat.
Smyth, J. M.— (1948) J. din. Invest, 21, 617.

Bryan, R. S. and

376

HyPERCITRICBMIA IN HUMAN CANCER
FACTORS CONCERNED IN PATHOGER^S ^ TREATiMENT

H. M. LEMON * J. H. MUELLER, t J. M. LOONEY
W. H. CHASEN AND MARCIA KELIMAN

From the Division of Neoplastic Disease, Department of Medicine, the Department of
Biochemistry, Boston University School of Medicine and the Outpatient Clinic, Boston
Veterans Administration, Massachusetts, U.S.A.

Received for publication January 12, 1960

Patients ivith leukemia, metastatic carcinoma and sarcoma possess altered
carbohydrate metabolism as shoum by reduced glucose tolerance (Marks and
Bishop, 1957), and elevated resting venous lactic acid (Cori and Cori, 1925).
Enzjunes concerned in tissue glycolysis are often increased in activity in serum
during neoplastic disease progression, including phosphohexose isomerase
(Bodansky, 19546), aldolase (Sibley Eleischer and Higgins, 1955), and lactic
dehydrogenase (Hill and Leid, 1954). Acid phosphomonoesterases hj’-drolyzing
tliree-carbon substrates produced during glycolysis are also increased in activity
in venous blood from patients ivith breast and prostate cancer (Reynolds, Lemon
and B 3 'Tnes, 1956). Observations indicating possible abnormalities of the ICrebs
tricarboxylic acid cycle in human cancer patients however are limited to increased
DPN-dependent dehydrogenase activity in sera of patients with hepatic meta-
stases (Wolfson, Spencer, Sterkel, and Williams-Asliman, 1958 ; Schwartz,
Greenberg, and Bodansky, 1959), and a single report of abnormal venous citric
acid (Kyle and Canary, 1957). In this communication we are reporting hyper-
citricemia as a frequent abnormality of untreated advanced cancer. A
preliminary account of these observations has been published elsewhere (Lemon,
Mueller, Looney, Chasen and Kelman, 1959).

METHODS

Citrate concentration in blood obtained from various sites has been determined
utilizing the Ettinger modification of the higlily specific pentobromoacetone
method (Ettinger, Goldbaum and Smith, 1952). Blood samples were obtained
from volunteers and patients folloudng a 12-hour fast and analyses performed in
duplicate in most instances. Cldoral hydrate which is the only Imown drug
possibly reacting to this proceedure has not been admimstered within 12 hours
to most of the patients or any of the volunteers studied. As a further check on
the methods used, citric and other organic acids have been separated mm
concentrated from sera by a method developed by Dr. H. H. Wotiz, using 4n KOH
to precipitate serum proteins followed by neutralization to pH 6-0 with concen-
trated HCl. The precipitate was centrifuged and washed once with distilled
HgO. The washings combined with the supernate were made slightly alkaline

‘Present address: Massachusetts Memorial Hospitals, 750 Harrison Avenue, Boston 18,

Massachusetts. , , -.c i

t Present address : National Naval Medical Center, Bethesda, Marjdand.

hypercitkicemlv in human cancer

377

with 2-5 N NaOH, and reacidified by bubbling COo tlirougli tlie solution. These
extracts were evaporated to dryness, resuspended in 1-0 c.c. ethanol and then
chromatographed, along Avith reference citrate standards (Stark, Goodbar, and
OAvens, 1951). The citrate zone AA'as then eluted, and the Ettinger procedure
applied to the extract. A close agreement AAoth the original analysis of unex-
tracted serum was usually obtained in sera from cancerous and non-cancerous
patients, indicating that citrate rather than some other organic acid Avas actually
being measured.'^

Blood was obtamed in some Amlunteers and patients by simultaneous posterior
iliac crest aspiration, arterial puncture and Amnipuncture. EolloAA'ing the initial
sample 0-5 g. of sodium citrate as a 4 per cent solution Aims injected intraAmnously
in a two minute period in many patients, Avitli additional Amnous samples obtained
at 5, 10, 20 and 30 minutes folloAA'ing commencement of injection. Since it Avas
soon found that citrate concentration decreased rapidlj"^ AA'ith time betAveen
5-30 minutes after injection, comparable to a first order reaction (Lemon, Mueller,
Looney, Chasen and Kelman, 1959), only 5 and 30 minute samples AAmre routinely
collected for measurement of clearance rates. Analysis of serum calcium (Clai'k
and Cobip, 1925), phosphorus (Eiske and SubbaroAV, 1925) and glucose before
and 30 minutes folloAAung injection Avas performed on a sample of 40 patients.
Periodic serum calcium, phosphorus and alkaline phosphatase (Bodansky, 1932)
analyses were performed in addition on most of the cancer patients.
Simultaneous obserAmtions of copper-resistant serum acid phosphatase, (Reynolds,
Lemon and Byrnes, 1956) and glutamic oxalacetic transaminase, (Franco, 1957)
Avere made on the same serum sample, in a representative series comprising many
of the cancer patients.

Patients Avere classified according to the nature of their principal disease.
Biopsy proof of cancer was obtained in all the cancer patients inAmstigated, and
the extent and location of their metastases was assessed from clinical and radio-
logic diagnostic procedures. Table I summarizes the diagnostic information on
the patients studied.

In the statistical evaluation of data, only the initial serum-observation was
utilized for the comparison of means between different groups of patients with
various ^seases. Tliis tends to underestimate the frequency of elevated blood
citric acid, wliich is more frequent in the more adA^anced stages of cancer, but is
necessary for a true comparison between cancer and other diseases, in which only
a single observation was available for each case. Hypercitricemia has been
e ^ serum citrate value exceeding tAvice the standard deviation of the mean

0 lea thy volunteers of the same sex, a value which is at the 95 per cent level

01 confidence for abnormality.

^■Citrate dynamics

residt of minutes ( A) Avas regarded as the

resuh of rapid dilution of the 0-5 g. dose into plasma and extraceLlar fluid, an

"I'olo lIoTw repeatedly yielded 95-98 per cent reooveiy of citrate added to

< ;M'liealeana\yscTfor'cltrar have been obtainTwhen

“'•dS). All SOTO wore the method reported by Saffron andDend^tem

fill within n few dnv frozen upon separation from clot and analyses should bp parr" a

-trntio., with prtn °1 sera may show 5-10 per ce^tTangS i^ citrate

of analysed. 8'='^ storage. Simultaneous citrate standards were routinefy run with each

378

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN

Table I. Classification of Clinical Matevial

Number

Diagnosis
Healthy volunteers

Rheumatoid and de-
generative artliritis

Source of material
Medical students. Hos-
pital personnel
Ambulator 3 ’ out-patients,
with 15-j'ear docu-
mented historj'

patients A^utrition

71 Excellent

32 Good

Stage of Disease

Stage II. m. American
Rheumatism Associa-
tion.

Non-cancor disorders .

Chieflj’ hospital patients,
some bed-fast

Good to fair

—

Pre-malignant lesions
and benign tumours

Ambulator^’ out-patients
and hospital in-patients

Excellent to
good

—

Carcinoma and sar-
coma

Ambulator}' and bed-fast
hospital patients

195

Good to fair

All stages, from early
localized surgically
cured to distant me-
tastases.

Total

—

358

—

assumption wliich appears valid (Bunker, Stetson, Coe, Grille, and Murphy, 1955).
Tills distribution space S was determined by dividing the total in fig. (0'5 X 10®)
by A given in fig. per ml. In healthy volunteers and cancer patients the mean
for this space was 21-6 liters for males and 14-0 liters for females (Table II).

Following the peak concentration of citrate 5 minutes after start of injection
serum citrate concentration rapidly declined in a semilogarithmic manner
compatible with a first order reaction, the initial baseline concentration verj'
nearty being attained in normal, artliritic and cancer patients at 30 minutes time
(Lemon, Mueller, Looney, Chasen and Kelman, 1959). The rate of decrease of
serum citrate concentration approximated 1 /ig./ml./min., which when multiplied
bj’^ S X 60 in each case provided an estimate of tlie hour!}’' rate of citrate clearance
from plasma and extracellular fluid, by diffusion, metabolism, and renal excretion.
In normal males, plasma clearance approximated 900 mg. per hour and in females,
800 mg. per hour. Urinarj^ citrate excretion was not measured, since preliminary
studies showed poor correlation between serum and urine citrate concentration.
Citrate excretion in the urine appears to fluctuate quite independently of serum
values owing to high renal uptake and metabolism (Herndon and Freeman, 1958).
Only a small fraction of plasma citrate passing tlmough the kidney is excreted,
the amount being affected by acid-base balance and vitamin D content of the
diet (Yarbo, 1956).

2. Fasting venous citrate concentrations and disease

In conformitj'’ with observations published by Rechenberger and Benndorf
while this study was underway (Rechenberger and Benndorf, 1956), the mean
venous citrate concentration of healthy females was found to exceed that of ma es
of comparable age groups (Fig. 1 ; Tables II, III). The mean fasting ''''Gnous
concentration of male and female patients %vith rheumatoid and osteoartnn is
was almost identical to that of their sex-matched volunteer controls.

Patients with benign tumours such as benign prostatic hjqjerplasia and marn-
mary dysplasia had citrate concentrations also within the normal range or leir

HYPERCITRICEMIA IX HUMAN CANCER

371 )

Table II. — Observadom of Citrate Dynamics in Hcaitiaj Vobintccrs anti
Patients with Benign Diseases

Moan

fnsting

I'roquoncy

venous

Distribution

Clcnrnnco

Number

citrnto

ob.scrvntioii

Hpaee

rate

Group

conccu-

ftbovo

(liltTK

(mg./br.

iS.E.)

Se.x

cases

trntion

Rnnge

2xS.D.

±S.E.)

Normal

Volunteers

. 27 .

27-2±l-3 .

lfi-4-

21-64-2-4 .

896 4- r, 8

(;j<0-001)»

45-7

(w = 0-0l2)*

. 44 .

38-3±l-8 .

••

14-94-1.3 .

7994-38

0 J .00/

0 — - ^0

Non-cancer

Hepatic cirrhosis

. 3 .

33-9

IS-

61 -1

4 .

40*5

34-8-

Osteo-porosis .

. 2

.51 -0
20-2-

Pregnancy

3 .

25-0

38-4

14-4-

Other disease

. 10 .

34 * odz 0 * < ,

31-0

14-S-

84-9

. .

. 10 .

39-li4-8 .

20-4-

Rbeumatoid arthritis
Osteo* and degenera-
tive arthritis

. 23 .

. 9 .

27-g±l-2 .
26-oi0-4 .

C4-4

20-30

23-5-

30-5

31-4i4-8 .
20-5±2-l .

1027 ±33
9.75±7I

3=4-0%

^re-malignant
Mammarj' dysplasia .

. 13 .

30-4±2-7 .

16-1-

Ojvecomastia .

. 2 .

49-9

24-0-

^^enign prostatic hy-
Pcrplasia

. 12 .

29-0±2-6 ,

58-5

1.5-7-

44-8

3 = 11-1%

Grand total .

. 163 .

. .

- 9=5-5% .

Significance between upper and lower figures.

«nd ^vH^clLTca%

concentrationMSble

’^ad fasUngfenL?ciSo^® ’«dth untreated metastatic carcinoma of the breast
!:>^«nteors%rtSc (P = < ' 01 ) in excess of

'Concentrations of venous cRrnJ mammary dysplasia (Tables II IV)

I’Jitientsn-ifb often exceeded more than .f; 7 7 , h

380

LEMON, MUELLER, LOONEY, CHASEN AND KBLJL4N

rarely in non-cancer patients, including one obese female with mild uncontrolled
diabetes melbtus, an anxious male wtli questionable peptic ulcer symptoms and
negative X-ray findings, and one case of osteogenesis imperfecta.

Eio. 1.— -Mean fasting serum citric acid concentration in healthy volunteer controls and
untreated metastatic breast cancer in females, grouped by age decade. Small numbers at
each point indicate number of patients observed. Horizontal dashed line indicates mean,
with range of standard error indicated by upright bracket at left, for the entire series of
patients in each of the three classifications.

• • Untreated metastatic breast cancer (2d).

Controls

O O Femalo (55).

□ □ Male (37).

The discrepancy between the total cases and the sums at each point in this chart

represents those rare cases for whom age was unknown from available records and
solitarj' cases of other age decade categories which were not suitable for plotting as
means.

3. Citrate clearance rate and disease

No significant sex difference was noted in mean citrate clearances (Table Hi-
Male patients with extensive rheumatoid arthritis cleared injected citrate from
their blood stream at rates which did not differ sig^cantly from male or female
normal volunteers. There was no si^’ficant deviation from volunteers in the
average rate of citrate clearance by patients with various cancers other than breast,
except for males with inactive post-therapy non-breast cancer, who showed a
subnormal mean clearance rate in the small group tested (Table III). However,
untreated patients with disseminated breast cancer tended to have the mg ms
mean rate of citrate clearance, of any of the groups studied, as well as the big es

HYPERCITBICEMIjV in nUAIAN CANCER

38 1

Table IlL-Obsenv/io,,^ ofCHralc Dytmmics in Palknls with Cancer

Group Sex

Hon-breast Cancer

Local, pre-operative . AI.

Inactive post-therap 3 ' JI.

F .

Active distant inetn- JI.

stases F •

Carcinoma of prostate 31.

Carcinoma of Breast

Local, pre-operative . F.

Inactive, post-therapy „

Active distant meta- „
stases no therapy

Active metastases „
treated by sex hor-
mones

Active metastases „
treated by cortisone
or prednisone

3Ienn fnsting
venous
citrate
(;ig/iT>l.
Number -I- S.E.

of initial

patients observnfion)

5 3G-3±5-G

7 3G-0±4-3

0 38-8±7-2

42 42-0±3-2

35 35-7±2-2

8 37-G±5-fl

107

G 27-8±2-7
20 3C-5+I-7

41 52-l±3-5

18 4G-2±5-G

I'roqiiericy ,

of oWrvii- Distrilmlion Climrnnco

(ions Number apnro rate

execetling of (lilors (ing./nr.

Range 2 X S-B- patients i S.E.) S.l'>.)

1.5-7-50 .1

72-5 1

ir)-!)-52-ft 2

1G-0-7G-1 3

1G-.5-100 14

12-1-G9-9 3

14-9-GG-r> 3

's 18-7±3-G 4tl0±107

(i Ifl..lj;4-I IOI3-J; 90

12 ir>-8-i;2-l 828^127

8 18-2i:2-0 7iriJ;I30

29=27%

20 -7-39 -.5 0
22-3-.51-4 0
19-4-llG-G n

18-2i2-7 992 ±72

22- 9-72- G

(p<0-01)*

19 - 4 - 93-0

20 1.5-r»±l-5 720± C9

lC=18-4%

Grand total

195

* Significance of difference between upper and lower figures.

45=23% 78

mean fasting venous citrate. T'ollowing therapj’’ ivitli adrenal corticoid hormones,
breast cancer patients showed a reduction of citrate clearance rates, as well as a
reduction in fasting citrate level.

In one patient with breast cancer, citrate clearance was simultaneously
measured in mixed venous blood, and in venous blood pas.sing tlirough the primary
tumour. The citrate clearance rate was identical in both areas, according to our
method of calculation (Table IV). This suggests that the difference in blood
citrate concentration in the two sites was not due to uptake of citrate by the
neoplasm.

These results would suggest increased citrate diffusion from some body tissue
accounting for the increased blood level, rather than reduced tissue utilization in
carcinomatosis. Bone which contains over 1 per cent citrate (dry Aveight), in
sourc^^^f'^ matrix (Dickens, 1941 ; Thunberg, 1953) is one of the more obvious
thern^^l release, but metastatic cancer and malignant tumour cells

f considerable amounts of citrate (Potter and Busch, 1950 ;
and Shapiro, 1956 ; Miller and Garruthers, 1950).

Gi^rate distribution space and disease

"hich noted in the mean distribution space of healthy volunteers,

atistically significant at a level of confidence (p = rb -012 ; Table II).

382

LEMON, MUELLEK, LOONEY, CHASEN AND KEL5IAN

Table Phosphatase Activity and Citrate Concentration, in Venous Bhnit

Draimng Breast Cancer Primary Site, Compared to Mixed Venom Shod

Date

Time

1/13/58

. Fasting

5 minutes after

citrate injection
30 minutes after

1/16/59

citrate injection
Fasting

7 minutes after

injection

10 minutes after

injection

26 minutes after

injection

29 minutes after

injection

Venous blood from left
breast primary tumor

Acid

Citrate

phosphatase

({iraoIe/JOO ml.

27-6

23-5

39-1

25-8

16-5

29-4

33-5

27-2

19-5

28-3

Mixed venous blood from
right anfeeubital vein

Acid

Citrate

phosphatase

(pg./ml.) (fimole/100 ral.

26-9

13-6

70-0

30-2

34-5

16-4

33-0

23-6

65 '5

18-8

35-5

24-5

Mean values

21-6 29-6

44-2 31-5

Citrate dtuinmies
I/I3/5S

Distribution

space

—

11-6 liters

Clearance

—

990 mg./hr.

rate

1/16/58

Distribution

29 "4 liters

13-7 liters

space

Clearance

1300mg./]ir.

1300 rog./lir.

rate

A sex difference was not seen however in either of two groups of non-breast cancer
patients, in whom the average male distribution space approached the female
value. Male rheumatoid artlwitic patients, on the other hand possessed a liigher
mean distribution space for citrate than healthy volunteers, and were significantly
different in this respect from all groups of male or female patients with active
metastatic cancer of breast as well as other types of cancer (p = < -012). These
results suggest that the tissues of patients with rheumatoid arthritis are more
widely and rapidly permeable to injected citrate resulting in a 5 minute post-
injection peak value which is considerably smaller than observed in patients with
cancer, or healthy volunteers, as a result of dilution. Tin's observation may be
related to the unusual propert}^ of citric acid in solubilizing pro-collagen (Jackson,
1957).

5. Variations in citrate concentration in blood obtained from various sites

Citric acid obviously is in a state of rapid flux in the blood, with removal rates
in the vicinity of 0-8-0-9 g./hr. which must be matched by release of citrate into
blood at an equivalent rate. In an effort to learn more about this phase of citrate
metabolism, venous samples were obtained from blood leaving a large mucoi
adenocarcinoma of the breast, and compared to mixed venous blood from an
antecubital vein ;

HYPERCITEICEMIA IN HUMAN CANCER

ABSTRACT

Case No. 1. This Patent, F. W., aged G5, Boston City Hospital No.
1630205, had a 11 X 15 X 15 cm. -untreated neoplasm of the left breast
reported as muein secreting adenocarcinoma by biop,sy. Two large
superficial veins -were noted draining the massiv^e primary lesion, passing
from the mid left thorax upward to anastomose with communicating
branches to the left internal mammary vein. Two citrate tolerance tests
were carried out, 1/13/58 and 1/16/58, before and after 30 mg. daily of
prednisone therapy, starting 1/14/58 (Table IV). A radical mastectomy
on the left was performed on 1 /20/58, with 8 negative lymph nodes found.
A right radical mastectomy was performed on 1 /31 /58 for a simultaneous
primary on the other side, measuring 1-0 x 1'4 X I'O cm., reported as
medullary and scirrhus carcinoma, also with negative lymph nodes.

The results demonstrated clearly a decreased citrate concentration in blood
leaving this particular cancer, although acid phosphatase activity, which is believed
to diffuse from malignant tumours, (Lemon, Davison, and Asimov, 1654), was
definitely increased. The latter obser\'ation confirms that we were examining a
blood sample in the tumour venous bed which differed significantly in its properties
from mixed antecubital venous blood, in all six samples. As a result of this
observation, some other major source than tumour tissue for citrate diffusion
into blood had to be postulated to explain the elevated serum citrate noted in
breast and other cancer patients. Simultaneous blood samples from iliac bone
marrow, brachial or radial artery, and antecubital vein were then obtained, in a
series of healthy volunteers, and cancer patients. A uniform decreasing gradient
of citrate concentration from marrow to artery to vein wa.s observed both in
representative normal individuals and in cancer patients (Table V).

Table V. — Relative Concentration of Citric Acid in Blood freym Various Sites

Group

Honjtiiy voluntccr.s .

Molnstntic cancer patients
(<> umJer treatment)

Mean citrate concentration

Arterial blood
(as % of

No.

Jlarrow blood

marrow

•Sex

(/rg./ml.)

concentration)

34-1

(4 cases)
(range 81-94)

45-0

(range 87-98)

Venous blood
(as % of
marrow
concentration)

(range 31-92)
80

(range G7-9.5)

6. Serum calcium and phosphorus concentration

calci.lm significantly change serum

30 iuhtuff n”criod”of or'^ concentration in a sample of 40 patients within
notoc Tld ^ of ob.seryatmn. Hypercalcemia above 12-0 mg per cent

384

lemon, MUELLER, LOONEY, CHASEN AND KEL3LAN

Table Yl.—Calcinm Phosphorus and Blood Sugar Observations

Group

Healthy volunteers

Bheurontoid artliritis
Osteo-artbritis

Metastatic carcinoma, breast
No therapj'

Same, during prednisone
fheraps^ :

Less than 6 months
More than G months .
Other metastatic carcinoma
No therapy

Other metastatic carcinoma
No therapy

Number

Sex cases

M. . 15

F- . 20

M. . 23

„ - 9

F. . 23

„ 10

. 8

M. . 17

F. . 9

Venous
calcium
(mg./IOO c.c.)

. 10-2±0-27

. 10-l±0-03

- 10-I±0-I5

9-74;0-31
. 10-2i0-48

- 10-l±0-52

. 10-0±0-34

• 10‘4i0-45

9-9±0'37

Venous
phosphate
(mg./IOO c.e.;

. 2-84;0-08
. 3-2±0-l9

. 3-Ii;0-12
. 2-7+0-lS
• 4 - 2±0

. 3-6±0-28
. 3-5±0-2I

. 3-8±0-0S

. 3-6±0-04

Fasting
venous
blood sugar
(mg./IOO C.C.)

. 87-8i0-33
. 97 i4-o

(4 cases)

. 9I-8i4-0
. 88-li2-8

the various groups is shown in Table W. After an initial group of cancer patients
had been sampled, serum calcium concentration was not ascertained except in
those who by virtue of extent of metastases or clinical symptoms were considered
likel 3 '' subjects for lij'percalcemia. In all these cases, no relationsliip was ascer-
tainable between the level of serum calcium, and citric acid (Fig. 2). In a patient
with a functional metastatic carcinoma of the parathyroid gland, elevated citric
acid accompam'ed hj'percalcemia onljr intermittent^.

Metastases of other carcinomas than breast, and sarcomas in both sexes,
although sometimes associated with hypercitricemia also failed to show an}*
correlation with serum calcium or phosphorus concentration (Fig. 2).

7. Hypercitricemia and site of metastases

The major sites of metastases were compiled in patients showing hypercitri-
cemia, which in some patients constituted at least two major areas (Table tTI).
Not onl}’^ was hypercitricemia noted in the absence of clinically demonstrable
metastasis, but it was noted in association with each of the major areas of
metastasis. Extensive osteolytic activity was present in several patients with
metastatic breast cancer, or multiple myeloma without hypercitricemia. How-
ever, these patients were receiving some tj'pe of antitumour therapj' at the time
of observation. Major hepatic involvement, which might affect citrate
metabolism, w^as present in onl}’^ one-fotufh of patients with hypercitricemia ;
and adrenal metastases were uncommon. All the major pathologic tjpes of
cancer thus far investigated maj’^ induce hypercitricemia sooner or later.

S. Hypercitricemia and cancer therapy

In our preliminarj^ report it was emphasized that hypercitricemia was encoun-
tered far more often in patients whose cancers had not received any recent anti-
tumour therapj’’ (Lemon, Mueller, Looney, Chasen and Kelman, 1959), A more
extensive anafysis bj'^ cases indicates a substantially similar picture, with le
liigliest frequency (55 per cent) of abnormal citrate values in patients prior to any

hypercitricemia in human cancer

:}sr)

' •c5no' frnm oHier sitcs Avcrc receiving radiation or analgesic drug t]icrn]).>

::r STJe"^f .*owe,i a

citrate concentration in their terminal stages of cancer, m spite of thcrapj
was obviously ineffective.

120

100

i 80

=J.

C
o

60!

•D
O

5 4o:

□ s*

° ••

O D*

• •

gt •S'
^0

□

J L

6 9 12 15

Serum calcium, mg. per cent.

Fio. 2.— Scatter diagrams of relationship between serum citrate and calcium concentration
m 47 patients with neoplastic disease. Upper normal citrate concentration for males
pg/ml., females 62 ;tg/ml. The highest serum calcium concentration was seen in a male
With a functioning metastatic parathjToid carcinoma.

# 21 g. Metastatic carcinoma of the breast.

O 9 ?, Non-breast metastatic carcinoma.

D 17 (^.Non-breast metastatic carcinoma.

0. Hypercitricemia and serum enzymatic hyperactivity

enzyme analysis was not extended to the arthritics or
litcr-vtire e project since ample data was available in the

horn our ” values for these disease parameters, and also

l!i.30\ ^ ° patients (Reynolds, Lemon and Byrnes

MoS in I>'’'=»P»atase ™ elevated above normal in vroS

majority of the untreated breast cancer patients, and tended to

3S6

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN

Table Vll.~Location of Principal Metastases in Patients toiih Hypercitricemia

Pathological Number

diagnosis of

of neoplasm patients

A. Sarcoma . . 3

B. Epidermoid and un- 13

differentiated car-
cinoma

C. Adenocarcinoma . 8

(non-breast)

D. Adenocarcinoma of 10

breast

E. Other g

Location of metastases

Local

recurrence

and/or

Lung,

pleura,

media-

Liver,

portal

lymphatic

stinium

area

Bone

Remarks

—

Adrenals ( 1 ).

Brain ( 1 ).

—

Brain (2).

No metastases in car-
cinoma of bladder
( 1 ), adenocarcinoma
of colon ( 2 ), renal
cell carcinoma (]),
basal cell ( 1 ).

Total patients ivith hj’- 45
percitrieem/a (initial
observations)

10 12 10
(22%) (27%) (22%)

9 (No metastases pre-
( 20 %) sent in 10 %).

Table VIII. — Relation of Anti-cancer Therapy to Occurrence of Feiiott^
Hypercitricemia in 101 Patients with Various Types of Cancer

Frequency of hypercitricemia*

Source of cancer
Breast

Other genito-urinary
Respiratory
Gastro-intestinal
Mesenchyunal .

No anti-cancer therapy
during observation

^ A ^

Total cases Elevated (*)

18 9

4 3

8 7

6 2

5 2

Anti-cancer therapy
conciurently' or within
past 6 months

, ^

Total cases Elevated (*)

28 11

13 5

11 4

3 2

5 1

Totals .

41 23

(55%)

60 23

(38%)

* > 2 X S.D. of normal mean venous citrate level (95 per cent level of conPdeuce).

increase with time, especially during the second six months of prednisone therapj'
(Table IX). Phosphohexose isomerase analyses showed a similar trend. The
data are insufficient to draw conclusions from either alkaline phosphatase or
transaminase measurements. Citrate concentration, however, decreased during
the &st six months of prednisone treatment of breast cancer, to values well
within the normal range, before a later secondary rise, suggesting diflFerent factors
influenced venous concentration of the latter during treatment compared to
various enzymatic functions. Other types of metastatic cancer in men and women
also often had abnormal acid phosphatase and phosphohexose iseromase activity
as shown by the mean values, although the variation of activity from case 0

HYl’EnCITRICEMIA IN HUMAN UANIHCH

Table IX.—Enzymc Adivtli/ of Fchom,<! Blood nml Cilratr ('oitrnilraiimi

Aikfilino
phos]>!intnso
(Bodnnsky
units)
Under ii

Copper

resistnnt

neid Phospholieso‘i<>

|)hospliiitnse isomerase

(piuolo phenol/ (Hodansky

100 ml.) units)

Under 24 pinole/ Under ■10
100 ml.

(•lutainie
oMdaei'l ie
transainiiuiue
(units)
Under 20

iitiilH

Metastatic carcinoma „
of breast
Xo therapy

Same, prednisone
therapy 6 months or

less

.Same, prednisone for
over R months
Other metastatic car- M.

cinoma
Xo therapy

Other metastio car- F.

cinoma
Xo therapy

Numbers in parentheses

5-95il-l.'$ S.E. 3S-Si-l-2 .S.E.

U-C

(5)

5-8±1-7

( 12 )

8-2±3-2

( 0 )

40-l±C-<)

( 1 . 3 )

46-S-J-4*9

( 10 )

33-3±5-0

(19)

.32-8J;4-f.

( 10 )

r>9-2-ilK-fi

(10)

f>3-3-i-.';2

(f)

ii-r.i.'i.fi

(14)

(10)

indicate number of patienl.s tested, upon u hieh mean £■

b'lml ili(;
eilrnle

eoneentrnlion
(py./inl.)
27-2 ; I -3
(27)

3M-3.i I-K
( 11 )

4.7-2-t 3-8 S.T:
(29)

33 -ti ; 2- 9
(13)"

•14 -3 1 r.-o
( 12 ) ■

>3-0 i 3-8

( 2 .',) "

39 -on r,-0
(1.7)

S.E. is based.

enzymatic abnormal itie.s ni.so njinDarctl
d aepenclently of citrate concentration in many individual case.s.

of oophorectomy in p.tict,
jWmng prednisone 2(^30 dadv 'r, ' suppre.ssion

breast cancer cases (Lemon f 959 ) \!Hn^r'' «dvancod

administration upL citrate'^d^Tm ' immediate effect.s

and in 10 cancer patfents^ A^^^^^ i"ade in 7 healthy

trate \vas noted in healthv vr.i f inconsistent change in fasting \mnoiis
patients nith hypercitricemia receiving prednisone, n'hile the cancer

toaot ?o"ar,lSf “ ™ so‘od i

initially Prednisone therapy a£i TvhQ^p^r.^f’f ^ nulUfied the anti-

* S‘SpSf SI

VO, sec *s

388

LEMON, MUELLER, LOONEY, CjEIASEN AND KELMAN

Table X.~Effect of Prednisone Therapij on Citrate Clearance
Volunteers and Cancer Patients

Per cent
change
from

Healthy

Duration

pre-therapy

volunteers

therapy

observation

(Male)

J. L—

3 days

+ 55

J. S—

+ 75

B. I_

-12

R. N—

-37

(Female)

L. K—

—11

R. D—

—36

V. D—

-62

(Dosage

= 30 mg. /day)

7 patients Jlean

= -4%
(7 tests)

Cancer

Duration

patients

Origin

therapy

(Male)

F. C—

Lung

8 days

(Female)

E. AV—

Breast

1 day

I. G—

Endometrium

27 days

H. A—

Breast

2 days

6 weeks

months

P. I—

»?

6 days

2 months

>»

5 months

6 months

B. L—

>»

9 days

B. T—

11 days

L.M—

„

9 months

„

1 year

A. M—

12 days

M. C—

6 months

M. L—

9 days

•t

2 weeks

11 patients

Per cent
change
from

pre-tliernpy

observation

-70

+31
-63
-13
+22
-41
-61
-43
+ 18
+31
-46
-67
+ 3
-24
-14
-90
—60
-22

Mean
= -28%
(18 tests)

HYPERCITRICEMIA IX HUMAX OAXCER

DISCUSSIOX

H\T)ercitriceniia in variows t-s^pes of cancer has not been frequently noted by
several previous observers (Schersten, 1931; Kottino, Hoirmnn and Brondolo
1952) vdtli a single undocumented exception (Kyle and Canary, 1 ho i ). Bevicu' ot
the case material utilized in several of these papers indicates that only a small
number of patients ■were studied, of rvliom many had already received anti -cancer
therapy. Furthermore, it is not clear whether fasting bloods were always
utilized for analyses, which will usually ydcld the peak venous citrate concentra-
tions. Kelatively few case reports are included of breast, prostate and lung
carcinomata which comprise the majority of our patients with hy])crcitricenna.
Simultaneous pre-selected control groups were not used in some previous studies,
retrospective controls being used. These differences appear to exjdain most, of
the discrepancies between our obsen'ations, and previous reports.

In spite of inflammatorj'' disease involving bone and joints in rheumatoid
arthritis and metabolic disorders, such as post-menopansc osteoporosis, serum
citrate was normal in all non-cancer cases. Atrophic bone changes were frecjuentl 3 ’’
widespread as shown bj'' X-raj’’ at the time of our sampling. Tliis suggests that
citrate measurement can be a useful adjunct in differential diagnosis of the benign
or malignant nature of some osseous lesions, in which demineralization i.s a
promiivent feature.

From our observations kypercitricemia appears to be a relative!}- common
disorder usually independent of hypercalcemia in patients with advancing neo-
plastic disease invading liver, lung or bone among otlier tissues. Hj’percitri-
cemia during active cancer growth is of particular interest in that it may represent
a dysfunction of the Krebs cycle through excessive citrate production or deficient
citrate utilization via condensation to acetoacetate, either in cancer or normal
tissues or both. Destruction of the activity of CoenzjTne II (TPN) might result
m such a disturbance. In addition citrate equilibrium in osseous tissues maj' be
disturbed, as seen in hyperparathyroidism or Paget’s disease wdth hjqoercitricemia
secondary to osteolysis (ICissin and Kreeger, 1954 ; Chang and Freeman, 19506).
n this latter case, a negative calcium balance and hypercalcemia might be
expected to accompany hypercitricemia, as we have observed in one case of
unc loning parathyroid carcinoma and in occasional mammary cancer patients,
th ^ ^ osseous destruction, although present roentgenologicallj’’ in many of

In^ iTorcitricemia breast cancer patients, was not severe enough to induce
contributoi^*^ ^ these patients and maj'^ be coincidental, rather than

fhe first to observe that parenteral administra-
tion in <sp marked hypercalcuria with minimal altera-

Frecman Their work has been confirmed (Chang and

attendant rZiU absence of hyperparathyroidism and its

^old aucment'i'i- ^sturbance hypercitricemia may produce a ten to twenty
concentration R calcium excretion without raising total serum calcium
in the ultra-fiUp of a marked increase

diminished °^d>Ia.sma calcium which is citrate bound, or because

‘''^•'crvations howp°’^ T of calcium citrate complex. These

''rnrcalcemia in „ ^ explain the rare coexistance of hypercitricemia and

™ one cancer population.

390

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN

Hormonal factors such as insulin (Pincus, Natelson and Lugovoy I94.))
epinephrine (Ihncus, Natelson and Lugovoy, 1951) and adrenocortical hormones
(1 incus, ISatelson and LugovojL 1951 ; Agrell, Lindell and Westlinc^, 1955) also
have been shmyn to alter venous citrate concentration. Hone of oiu lijmercitri-
cemic cancer patients ivas diabetic or receiving insulin, nor did we encounter any
pheoclmomocytomas in our series of cases. Although adrenocortical insufficiency
remilts in hj^iercitricemia in man (Martenesson, 1949), none of our patients was
suffering from acute adrenocortical insufficiency at the time of our studies, and
only one patient with leiom3’'osarcoma metastatic to the adrenal glands was
receiving adrenal steroid therapy, as a result of a previous Addisonian crisis. At
the time of our study this patient was in electrolyte balance. Although the
kidnej^s serve as a major site of citrate uptake from blood, significant impairment
of renal function was not present in our hypereitricemia patients. Hepatic
insufficiencj' was also absent in most h3^ercitricemia patients, including those
with hepatic metastases. Ho evidence was obtained that hj^iercitriceiiiia was
related to an3>' reduction of citrate uptake from blood (Table III).

The strilcing reduction of venous citrate concentrations to normal in previously
h5'percitricemia breast cancer patients receiving cortisone or prednisone, occurred
in spite of a 28 per cent reduction in clearance rate for injected citrate. Tliis
depression of citrate uptake from blood by adrenal steroid therap3" in all likeli-
hood is closely related to the reduction of acetate utilization which has also been
reported. Hennes and Shreeve administered doses of prednisone identical to
those we have utilized, to patients receiving ^^C-labelled acetate, and noted an
initial reduction of 20-30 per cent in rate of radiocarbon excretion compared to

control observations (Hennes and Shreeve, 1959). Over a 24 hour period a 10-15
per cent reduction of cumulative radioactivit3’^ excretion was demonstrated.
Hemieman and Bunker have reported elevated venous lactate and p3n’uvate
concentrations following adrenal steroid therap3'’ and in Cushings’ s3mdrome
(Henneman and Bunker, 1957), suggesting a decrease in p3muvate oxidation under
these circumstances. Impairment of glucose tolerance has long been recognized
as one of the most dependable laboratory manifestations of adrenal cortical

h5^erfunction.

The control of h3q3ercitricemia b3’^ adrenal steroid therap3'^ must be accounted
for b3’^ reduced citrate diffusion into blood from some tissue source. Hot ord}'
must this diffusion rate be ver3^ high prior to therapy to induce hj^ercitricemia,
in view of the 20-25 g./day capacity of the body to utilize citrate, but steroid
induced inhibition of diffusion from tliis tissue source must far exceed the net
overall reduction in uptake of citrate caused b3' predmsone therap3'^. nr
observations indicate that a major source of citrate enrichment of blood exists m
the sinusoids of bone marrow, wffiere at all times citrate concentratimi excee s
that of mixed peripheral arterial or venous blood. Adrenal steroid therap}
appears to have a more marked and consistent effect reducing venous cffra e 0
breast cancer patients than that of healthy volunteers. However, drflerences
in the duration and intensitj'’ of therapy or in the degree of sex hormone m u 1 m
so induced, ma}^ account for this variation in response. The ^eater prera ei
of hymeroitricemia in advanced breast, prostate and lung carcinoma pa len s j
whom osseous metastases are so common (Table III) and the infrequenc}
occurrence in bem’gn tumors or localized breast cancer suggest that
cemia is potentiated by widespread neoplastic invasion of bone marrov.

HYPERCITRICEMIA IX HUMAX CAXCER

391

simultaneous elevation of venous acid phosphatase in hyperc.tacemic patients
aho supports W marroiv invasion as the cWef souree for excessive diffusion of
citote^into blood, since this enzyme is elevated m venous blood in /o per cent
of natiSs ivith osseous metastases of breast or prostate carcmoma prior to
^i Japy (Reynolds. Lemon and Byrnes, 1956). We have a so found that iiiarroiv
sinusSd blood is far higher in acid phosphatase activity, than peri^ieral arterial
or venous blood (Reynolds, Lemon, Kaplan, Idelson, lilueller and Derou, 19o9).
Likewise, abnormal venous phosphohexose isomerase activity is often present m
mamman' carcinoma with osseous invasion (Bodansky, 19o4«), and elevated
alkaline phosphatase activity is ivell known to result from osseous or hepatic

invasion bj' tumour. . , , c

Since the early reports of Dickens and others concermng the presence^ ot

citrate in the organic matrix of vertebrate bone (Dickens, 1941 ; Tliunberg, 1953),
which contains 95 per cent of total bodj'’ citrate, a great deal of uork has been
carried out showing a close relationship between calcium and citrate metabolism
in response to various stimuli, and a current h^’pothesis of bone formation include.s
precipitation of calcium citrate on the superficial lamellae of bone trabeculae
(Neumann and Neumann, 1958). Enzjmies necessary for local production of
citrate have been described in osteoid tissue (Dixon and Perkins, 19.52). One
observed case of osteogenesis imperfecta, in a 5 year old boy, had a venous citrate
of 89-5 /fg./ml. prior to therapy, falling to a normal value after testosterone
therapjf had induced some calcification and clinical improvement. The move-
ment of calcium and citrate in and out of bone under the influence of parathormone
or lutamin D therapy is generallj' in the same direction (Carlson and Hollunger,
1954 ; Elliott and Ereeman, 1956), and a similar trend is apparent in our data.
IVith the exception of prostatic carcinoma which rarelj’^ induces h 5 ’^percalcemia or
Iij'percalcuria, cancer frequently resulting in hjqDercitricemia such as lung or
breast are also prone to develop hjqiercalcemia. When the latter develops,
hjqwrcitricemia usually co-exists. The Idiopathic " hjqrercalcemia reported in
advanced lung, breast, ovarian or renal cancer cases in the absence of detectable
osseous metastases may be possibly associated with h 3 ^pereitricemia, if the latter
abnormality were to be looked for (Plimpton and Gellhorn, 1956). A calcium-
binding sub.stance has been po.stulated in those cases in whom an elevated serum
alkaline phosphatase suggested bone disease.

Normal prostatic and mammarj^ epithelium secrete extremely high concentra-
tions of citric acid into their respective secretions as a result of specific sex
hormonal slimulation (Ilann, 1954 ; Lemier, 1934) and tlris function po.ssibly is
prcs(‘rvcd m soine endocrine dependent cancers. Talalay and Williams-Ashraan
Jmvc postulated that e.strogemc cellular stimulation is mediated via effects upon
tl.c balance of pjTidine nucleotide dependent transdehydrogenase systems
.nv., Ivrtl u,c ottte Kxete cjrte (Talaky and WillLa?ia„^9™|

uluch vould therefore govern the production and/or utilization of citrate bv
hormone sensitive tissues. cniare ny

Our one negative observation of citrate diffusion from breast t

in a jialient with jirimarv disease lacking demonstrable Ivmobpt' ^^rred

inetastase.s and whose venous blood levels w^Tumm^^^ o’”
marrow served as a maior source for the bi'frb r.:f + ’ Wmor cells in bone

oMeolytie bone destruction might be explainS ^ ®

392

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN

the diseased sinusoids to compete mth phosphate for the calcium In^droxyapatite
(ISeumann and Neumann, 1958). Neoplastic invasion of soft tissues may also be
lacnitated by citrate induced solvation of procollagen (Jackson, 1957)

Numerous observations attest to the striking ability of cortisone and pred-
nisone therapy to control hj'percalcemia in cancer, and to induce recalcification
of osseous metastases in breast cancer, coincidental with reduction of circulating
citrate concentration to normal (Lemon, 1956, 1957, 1959 ; Nissen-Meyer, 1957)
Adrenal corticoids have been shoma to antagonize the action of estrogens upon
hormone-dependent target tissues such as endometrium, nullifying both water
inhibition and groudh (Szego and Roberts, 1953 ; Huggins and Jenson, 1955 ;
Velardo, Hisaw and Sever, 1956) as well as exerting a depressing influence upon
the growth of a number of different t3'pes of transplantable and spontaneous
cancers (Pearson, Li, MacLean, Lipsett, and West, 1955 ; Rusch, 1956). As a
working hj^iothesis it may be postulated that the normalization of venous citrate
and subsequent recalcification of some osseous metastases may also represent the
result of direct inhibitorj'^ effects exerted upon osseous mammary carcinoma
metastases as well as indirect effects secondary to depressed sex hormone secretion.
Our data is insufficient as yet concerning the influence of steroid therap3' upon
h3q)ercitricemia in other types of metastatic cancer to draw further inferences.
Although clinicall3' obvious metastases were noted in hepatic or pHlmonar3^ or
other areas without radiologicall3' detectable bone involvement in 71 per cent of
our h3q)ercitricemia cases v-ith metastatic cancer (Table VII) the prevalence of
circulating tumour emboli in over 50 per cent of patients with malignant neo-
plasms insures that the sinusoids of bone marrow become the repositor3' of
active or inactive metastases, sooner or later in most forms of advanced cancer
(Fisher and Turnbull, 1955: Moore, Sandberg and Schubarg, 1957 ; Engell,
1955).

Finalty, h3q)ercitricemia to the levels which we have observed must produce
some major disturbances of membrane perraeabilit3’', resulting from alteration of
the mono-valent to di-valent cation ratio tlnrough calcium binding by elevated
citrate. Normall3'' female venous blood contains about 0-6 m-equiv./I. of citrate
(38 /ig.jml.) capable of binding a similar amount of ionized calcium (Hastings,
MacLean, Eichelberger, Hall and Da Costa, 1934). About 65 per cent of serum
calcium is normally ultra-filterable or 3-24 m-equiv./l. (Neumann and Neumann,
1958). Bicarbonate, phosphate and citrate are the principal anions available to
bind diffusible calcium. Under normal circumstances, ionized calcium averages
about 2-66 m-equiv./l., so that citrate bound serum calcium constitutes a labile
fraction amounting to nearl3r one-sixth of total diffusible calcium. If citrate
concentration rises to the rmlues herein reported, then 1-0-1-S m-equiv./l. or more
of sei-um calcium ion might be complexed with citrate, assuming no slult ol le
latter from protein, leaving only an estimated 1-2 m-equiv./l. of ionized calcium
for control of membrane permeability. Hastings and co-workers cleariy sliowed
that calcium citrate was metabolicalty inert in so far as the frog rear u as con
cerned, up to values as high as 20 m-mole/ 1 . (Hastings, MacLean Eichelberger,
Hall and Da Costa, 1934), and hence cannot function m the control of membrane

^^™ontrary to some earlier reports (Allen, Clark, Thornton
h3q)ercitricemia has been observed as a hazardous coraphcatio - g

transfusion in infants (Wexler, Pincus, Natelson and Lugo\ 03 , ■ ),

HYPER CITEICE^IIA IX HIIMAX CAXCER

393

massive blood replacement therapy in adults rrith hepatic cirrhosis and Weeing
varices major vascular surgery and major operations m the portal area
Stetson Coe, Grillo and Murphy, 1955). Bunlcer and co-morkers observed that
the immediate rise in serum citrate after infusion of varjnng amounts of citrated
blood varied vith the rate of administration and total amount of blood loused.
Somewhat higher arterial blood levels were obser\md m patients wnth hepatic
disease, than in other pre-operative patients under pre-medication. Values were
noted as high as 300-400 //g./ml. in their senes (± O-0-6-5 m-equiv./l.). Depres-
sion of ionic calcium concentration calculated on the basis of total calciuna
total protein and citrate concentration (Hastings, MacLean, Eichelberger, Hall
and Da Costa, 1934) to values as low as 1-10-1-40 m-equiv./l. was associated mth
hjmotension or shock in the majority of cases, a few of whom responded to
intravenous calcium ion therapy. Only rarely did their patients show tetanic
manifestations wliich we have not obsenmd as yet, either. With calculated
ionic calcium above 1-50 m-equiv./l., no hypotensive phenomena were obsenmd.
Less deleterious subjective effects may result from smaller elevations of serum
citrate such as we have noted, which consisted primarily of anorexia and nausea,
and emphasize that hjqiercitricemia may contribute to malaise of the cancerous
patient. Eeduction of blood citrate levels to normal by prednisone therapy,
and the simultaneous striking subjective improvement of many patients, including
their strength and appetite, may be related to a restoration of a more normal
ionic cellular environment.

In most of the adult surgical patients in whom massive replacement of blood
is iiecessar}’^, extreme activation of the pituitarj’’-adrenocortical system by the
underlying catastrophic disease probably contributed to hjq)ercitricemia, by
impairing the metaboUsm of exogenous citrate, such as we have observed occurs
after adrenal cortical steroid therapy.

Cookson and co-workers have suggested that hypothermia which has been
utilized in vascular surgery may compound the hazard by further reduction of
ability to utilize large amounts of administered citrate (Cookson, Costas-Duiieux
and Bailey, 1954). Anoxia per se may also induce hj-percitricemia (Hallman and
Forsander, 1952).

Bunker and co-workers obtained fairly satisfactorj" agreement between the
observed increment in arterial citrate concentration after infusion (average in
11 paticnts=13-7 mg./lOO c.c.) and the increment predicted on the basis of
equilibration in extracellular fluid within 6-16 minutes (averaffe=ll-2 mg /lOO
c.c.) If the latter figure is corrected for the mean rate of fall we have noted in
seniin citrate concentration after termination of infusion, approximately 1 «g /
in ./inimite in both sexes m the period 5-30 minutes after injection, the expected
citrate concentration would be 12-5 mg./lOO c.c. from their data, on the assump-

t I ^ T clearance process begins immediately with initial elevation of serum
Cl r.i e. IS lively that the amount of protein bound calcium was overestimated
n-oiii the total protein concentrations observed in manv nf tv J

eridera^e' hitcai^ ^ a

„'f citrS

28 S

394

even with
1957).

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN
advanced liver disease (Howland, Bellville, Zucker, Boyan and Cliffton,

SUM5IARY

Citric acid concentration has been measured using the pentobromoacetone
procedure in blood from bone marrow, arterial and venous sites, in 358 healthy
volunteers, arthritics and patients with various types of cancer in aU stages. The
clearance of exogenous citrate from blood has been estimated in a representative
fraction of each group of patients and approximates 0-9 g. per hour. Women have
been found to have consistently higher fasting venous serum citric acid than men,
vdth a slightly lower citrate clearance rate. Bone marrow blood consistently
shows the liighest concentration of citrate, followed by arterial and finally venous
blood. Venous citric acid concentrations greater than twice the S.D. from the
mean for each sex {“ hj^ercitricemia ”) occurred in 4-2 per cent of healthy
volunteers, in 4-6 per cent of all patients vdth benign diseases, in 11-1 per cent ol
patients vdth pre-malignant tumors, and in 23-6 per cent of patients with jancer.
Anorexia and weakness were generall5'' noted in the latter group. Hj'percalcemia
exceeding 12 mg. per cent (6 m-equiv./l.) was noted in only 3 patients in the
entire series, but abnormal acid phosphatase and lactic dehydrogenase activity
usually accompanied hjq)ercitricemia. Advanced carcinomas of the breast,
prostate, and lung were most commonly associated with hj'percitricemia, probably
as a result of osseous metastases. Prednisone therapy’^ in advanced mammary'
carcinoma usually reduced elevated blood citric acid to normal simultaneously
reducmg by 28 per cent the utilization of administered citrate, indicating the
probable suppression of tumor grou'th in osseous and perhaps in other metastatic
sites.

These studies have been supported by a research grant jfrom the National
Cancer Institute, by the Rebecca Rice Memorial Grant of the American Cancer
Society to Boston University, by a grant from the Quincy United Fund, of
Quincy, Massachusetts, and a student research fellowship from the Massachusetts
Artlnitis and Rheumatism Foundation.

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824.

Bodansky, a. — (1932) J. biol. Ghem., 99, 197

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Bunker, J. P., Stetson, J. B., Coe, R. C., Gkillo, H. C. and Murphy, H. D.~(19oo)
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Carlson, A. and Hollunger, G. — (1954) Acta physiol, scand., 31, 31a
Carr C. W.— (1955) in ‘ Electrochemistry in Biology and Medicme , ShedJovsk}', i -
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Chang, T. S. and Freeman, S.— (1950a) Amer. J. Physiol, 160, 330.— (19o06) Ibid.,

160.341. . ^

Clark, E. P. and Collip, J. B.— (1925) J. biol. CSem 63 461.

CooKSON, B. A., Costas-Durieux, j. and Bailey', C. P. — (19o4) Ann. Surg., 139,
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HYPEBCITBICEJiIIA IX HUMAX CAXCER

395

Dickens, F.— (1941) BiocUm. J 35, 1101. .n -ok

Dieteich, L. a. and Shapiro, D. M.— (1956) Cancer Fe-s., 16 oSo.

Dixon, T. F. and Peekiss, H. R.— (19o 2) Bioc?icm, J., ^2, -GO.

Elliott, J. R. and Freeman, S. — (1956) Endocrinology, 59, _00.

Engell, H. C.— (1955) Ac(a cAir. ficand., Supp. 201, 1. .

eJSSe, R. H. Goldeah.1, L. R., S>hth, L H.-(1952) J bio . 199. '^^l.

Fisher, E. R. and Tuenbhhl, R. B.— (1955) Surg. Gynec Obsjel., 100, 102.

Fiske C. H. and Shbbabo'W, Y. — (1925) J. tio?. C^e?n., 66. 3/5. „ „ ,t -f,-

j.R^-co. — (1957) ‘ Sigmo-Franco Procedure ’, Sigma Chem. Co. Tech. Bulletin, Ro. oOo.
Gomori, G. and Ghlyas, E.— (1944) Proc. Soc. exp. Biol N.Y. 56, 226.

Hastings, A. B., MacLean, F. C., Eichelberger, L., Hall, J. L. and Da Go.sta, E.
— (1934) J. biol. Ghem., 107, 351.

Hallman, H. and Forsander, 0.— (1952) Ann. Med. exp. Penn., 30, 287.

Henneman, D. H. and Bunker, J. P.— (1957) Amer. J. Med., 23, 34.

Hennes, a. R. and Shreeit:, W. W.— (19.59) Proc. Soc. exp. Biol. E .Y ., 100, 246.
Herndon, P>.. F. and Freeman, S. — (1958) Amer. .J. Physiol., 192, 369.

Hill, B. R. and LE^^, C. — (1954) Cancer Res., 14, 513.

Howland, W. S., Bellville, J. W., Zccker, M. B., Boyan, P. /

(1957) Surg. Gynec. Obslet., 105, 529.

Huggins, C. and Jenson, E. V. — (1955) •/. exp. Med., 102, 347.

Jackson, D. S.— (1957) ‘ Connective Tis.sue. A Symposium ’. Tunbridge et al. (ed.).

Oxford (Blackv'ell Scientific Publications), p. 62.

Kissin, B. and Kreeger, N. — (1954) Amer. J. med. Sci., 228, 301.

Kyle, L. H. and Canary, J. J. — (1957) J. Lab. din. Med., 49, 590.

Lemon, H. M. — (1956) Clin. Congr. Amer. Coll. Surg., 6, 415. — (1957) Annals intern.

Med., 46, 457. — (1959) Cancer, 12, 93.

Idem, Davison, M. D. and Asimov, I. — (1954) Ibid., 7, 92.

Idem, Mueller, J. H„ Looney, J. M. Chasen, W. H. and ELelman, M. — (1959) Boston
med. quart., 10, 1.

Lenner, a. — (1934) Acta obstet. gynec. scand., Supp. 1 ; 14, 1.

jNLann, T. — (1954) ‘ The Biochemistrj.- of Semen ’. London (Methuen and Co., Ltd )

p. 16.

JIarks, P. a. and Bishop, J. S.— (1957) .7. din. Invest., 36, 254.

^Lvrtensson, j.— (1949) Ada med. scand., 134, 61.

Miller, H. and Carruthebs, C.— (1950) Cancer Res., 10, 636.

Moore, G. E., Sandberg, A. and Schubakg, J. R. — (1957) Ann. Surg 146 580
Neumann, W F. ^-d Neumann, M. W.-(1958) ‘The Chemical D3mamics of Bone
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Nissen-Meyer, R,— (1957) Acta endocr., Supp. 31, p. 314.

PLiMmoN, C. H. AND Gellhorn, A. — {1956) Amer T Merl 91 'rein
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.Sa„;,.sT,:K, li._(l!ni) Shad. Ard,] PhSfk. ti. ’

396

LEMON, MUELLER, LOONEY, CHASEN AND KELMAN

SCHAVAETZ, M. K., Geeenbeeg, E. aed Bodansky, 0— (1959) Proc. Amer. Ass. Caimr
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Velabdo, J. T., Hisaaa’, E. L,, Bever, A. T. — (1956) Endocrinology, 59, 165.
Wexler, I. B., PiNcus, J. B., Eatelson, S. and Lugoady, I. K. — (1949) J. din.
Invest., 28, 474.

WoLFSON, S. K., Spencer, J. A., Sterkel, R. L. and WiLLiAjM-AsmiAN, H, G.-— (1958)
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BRITISH JOURNAL OF CANCER

VOL. XIV SEPTEMBER, 1960 NO. 3

ON THE RELATIONS BETWEEN ATMOSPHERIC POLLUTION IN
URBAN AND RURAL LOCALITIES AND MORTALITY PROM
CANCER, BRONCHITIS AND PNEIBIONIA, WITH PARTICTJLAR
REFERENCE TO 3 : 4 BENZOPYRENE, BERYLLIUM. MOLY^B-
DENUM, VANADimi AND ARSENIC

PERCY STOCKS
From Colwyn Bay, North Wales

Received for publication July 12, I960

History and purpose of study

In 1954 arrangements were made jointly with the British Empire Cancer Cam-
paign, Jledical Research Council and Department of Scientific and Industrial
Research to commence an investigation into the amounts of smoke, certain polv-

elements in the atmosphere at a number of nlaces
and Liverpool Hospital Region with a view to relating them with
the mortality from Cancer m the areas concerned. In the first instance 1 0 instru-
ments for continuous collection of smoke on filter papers were nlacpcl in urban i

tunl sitaattaB. Theae were equipped .vith meterfr«re tta XA of
passing through, and the filter papers were changed at weekly or monthlv int^ T
sent for hydrocarbon analysis to the M R C Groun at St Ra ib

;4.oS STaVcLfc, ta Norfh Water,

notor garages, and one in an office each ndth nn r, f n *'^"0 Rt bus and

S il rS'gt''" T s»bseque’nt series were mad^by"'
liifrb.o, I ■ tbe Dunn Laboratorv'. The fi]t,ero ’ ’ of

"istruments working alongside the low snani + ^^companied by

l<m 10 1 * M hydrocarbon data from the f elements. A

mortah V "-ith tentaSe October

398

P. STOCKS

pkces in Lancashire (including one in a smokeless of c,; i • ,

West Riding of Yorkshire and at four sites in a large steelworks and^inoVsuJ 1
Alters at three places in lyneside conurbation. furSf J totai rfTlT.
locations as specified in Table I.

Table I.— Localisation of 41 Instruments to Collect Smoke

County or
conurbation
Anglesey .
Caernarvon
Merioneth*
Denbighshire

Flintshire .
Merseyside
conurbation

Cheshire .

S.E. Lancashire
conurbation
Lancashire

West Yorkshire
conurbation

IVest Riding
Tvnside conurbation

Samples for Analysis.

Representative
locations
Llangefni
Conway Vallej'
Blaenau Ffestiniog*
Ruthin
Wrexham
Flint

Birkenhead

Bootle

Liverpool, Princes
Road
Chester
Tattenhall

Salford, Regent Rond

St. Helens

Warringtonf

Ormskirk

Danvent

Burnleyt

Lancaster^

LeedsJ
Keigldeyf
Ellandt
Yorkt
Wetlierbyt
RiponJ
Goteshendt*
Newcastle-on-Tynet
(Whamcliffe St.)*

Alternative sites
in conurbations

Seaside (Hoylake)
RiversideJ
Roe Street:!

Edge LaneJ

Smokeless zonef

Shields RoadJ*

Special

locations

4 in steelworks.!

2 in Mersey tunnel.!

Bus depot (Edge Lane).}
Motor car garage-!

Office interior.*

* No trace element analyses. ! No hydrocarbon analyses.

! Analyses of 7 hydrocarbons were made here, and of 4 hydrocarbons elsewhere.

The present paper deals with the representative locations, which alone can be
related to mortality rates. Hojdake has not been included with these but con-
sidered as a special Merseyside site because many of the residents travel to Liver-
pool or Birkenhead to work or have retired after working there, and their death
rates cannot be fairly related to the local atmospheric conditions. The results
from the alternative and special sites have been reported upon in a separate paper
(Stocks, Commins and Aubrey, 1960). Data of total smoke and 4 polycyclic
hydrocarbons are available at each of the 26 places in the first column of Table I,
and amounts of trace elements at 23 of them.

A previous study was made also of the amounts of undissolved deposit and of
smoke in those count}’’ boroughs of England and Wales and administrative areas
of Lancashire and the West Riding of Yorkshire where measurements had been
recorded by the D.S.I.R., relating these with standardized mortality ratios for
cancer and bronchitis (Stocks, 1959). This has now been extended by addition of
more towns, using a more adequate social index, and a similar study has been
made of Greater London.

ATMOSPHERIC POLLUTION AKD MORTALITY

The main purposes of this paper are (1) to estabUsh more fomly the association
TiPtween smoke and mortality from lung cancer and respiratory disease, and (-)
to ascertain which of the polycyclic hydrocarbons and which of the trace elemen s

are concerned in this.

Evaluation of smoke and 'polycyclic hydrocarbons in air

The amount of smoke per cubic metre of air is estimated from a direct photo-
metric measure of the blackness of the circular portion of knonm area of the hlter
naner through which a measured volume of air has been drawn by a pump opiat-
ing continuously for a suitable period. At the end of the period, which may be a
week in a highly urbanised district or a month in a rural district, the paper is
changed and the results from all the filter papers throughout a year are aggregated
when an annual average is required. The blackness readings are converted^ to
milligrams of smoke by means of a standard calibration curve. The cahbration
was originally earned out on London smoke and it has been found to be reasonabl^^
satisfactory for smoke in other large tovms in Great Britain. Persistent errors in
the absolute values of the smoke concentration determined in this way would not
affect comparisons between districts, but there may be local variations in the
“ blackness ” per unit weight of smoke. The standard calibration applies to the
usual mixture of domestic smoke plus a little industrial and traffic smoke found
in the general atmosphere of British to\vns and it is unlikely to hold good for
special locations where industrial or traffic smoke predominate, such as road
tunnels. Any variations of this kind would be liable to reduce the correlation
coefficients between smoke and mortality.

The polycyclic hydrocarbons are determined quantitatively and a direct
measure of their concentration in the air is obtained by dividing by the volume of
air sampled in each case. They can also be expressed as a proportion of the
total amount of suspended matter (smoke) as in Table III, and this may be of
some importance, quite apart from the absolute concentration in air, but such
figures are subject to the errors mentioned above which affect the denominators

of the proportions. Sources of error which might depress correlations between
amounts of hydrocarbons and mortality rates or social factors include analytical
errors, instability of some of the substances leading to partial loss by complex
processes such as oxidation or evaporation during the interval between collection
of a sample and its analysis, and faults in obtaining representative samples bv
the choice of sites for the filters. Concerning the analytical technique a modi-
fication of this was introduced (Coramins, 1958) and applied in 1958-59, but all
data used m this paper from previous years have been corrected to allow for the
effects of this. As to stability, pjTene and fluoranthene are less stable than

avoriipc corrohtion between pjTene’and the other 1 f h «n(i the

n.« for the tnore .,„h.e be„eo„wene. It SXgt

400

P. STOCKS

of pyrene m the saniples must Jiave been so uniform as not to affect correlations
with other factors appreciably. ^

Faltdihj of correlations between smoke measurements and mortality rates

A source of difficulty in attempts to correlate these factors is that neither is
constant in time, and exposure to air pollution for a number of years may be
necessary to produce serious respiratory disease or initiate cancer. In order to
obtain meaningful rates from specific diseases in separate districts deaths throiwh
a period of years have to be aggregated and the rates standardized to allow For
differing age distributions in the populations concerned. Changes in the inter-
national rules for classifying deatlis to their underlying cause from 1950 onwards,
and changes in the rules for residence allocation of persons d 3 dng from ciironic
diseases after a long time in hospital which took effect in 1954, made it advisable
to use the period 1950-54 for lung cancer and 1950-53 for the other disease
groups in this study. The smoke measurements were made for the most part
during 1955-58. It has to be assumed, therefore, that although the amount and
nature of air pollution b 3 ’^ smoke ma 3 ' haAm changed in the last 10 or 20 5 ’-e 3 rs, the
relative changes haAm been the same in all the areas concerned, in Avhich case the
correlations AA’ith other factors Avould not be affected. Departures from this
assumed constanc5^ of relatiAm change Avould tend almost certainly to reduce the
true correlations and the obserA’’ed coefficients can safel3' be regarded as minimal
figures. This difficulty must arise in all attempts to relate mortalit 3 ' from chronic
diseases Avith measures of air pollution of AA'hich records exist onl5" for recent
3’’ears, and action Avhich has noAi' started to reduce such pollution in the worst
areas makes it an urgent matter to make such epidemiological studies before the
opportunit 3 ^ has gone.

Another possibly important source of error was the reliance on a single instru-
ment site to measure the aA^erage air conditions to Avhich the population of the
district surrounding it Avas exposed in the course of a year. A stud 3 '’ of this
problem has been made in Sheffield, Avhere it was found that Avhen an 3 '’ 8 instru-
ments Avere chosen to assess the aA^erage smoke concentration to AAffiich the AAdiole
of the population in the city Avas being exposed, the mean of the 8 had a coefficient
of Amriation of about 10 per cent (Clifton et al., 1959). This variation increased
in the inverse ratio of the square root of the number of filter sites, reaching about
28 per cent for a single site. Sheffield is peculiar in having nothin its area of
62 square miles altitudes aboAm sea IcAml ranging from 100 to 1400 feet and den-
sities of population in separate Avards ranging from 2 to 62 per acre, and the coeffi-
cient of variation is undoubtedly higher than aa'ouW be found in most cities. In
LiA'erpool AAffiere the river is a disturbing factor, smoke concentrations at three
locations AAmre 179, 215 and 204 in the spring-summer half and 500, 624, 604 in
autumn-AAunter, measured in mg. /1 000 m®. In small tOAAUVs Amriation AA'ould
generally be less, and in the 26 areas used in the present study the coefficient of
A’’ariation for a single site can reasonably be taken as 20 per cent.

In order to find what effect such errors could have on the correlation coem-
cients AAuth mortality seA^eral tests AA'^ere made on the lung cancer figures, assuming
the root mean square error of the smoke readings to be 20 per cent of the true
EAWges in the area. The 26 ratios (F) between the true average smoke value m
a district and the smoke reading at a single location in it Avere presumed to haA'e a
normal distribution AAuth standard deviation 0-20 and the3’' Avere first arranged in

atmospheric pollution and mortality

401

Irde Avere applied to the areas arranged in order of their lung cancer ^he

result was 0-82. It is to be expected, however, that the magnitude of the erro ,
Sardless of its sign, would in general increase with the size of the district pro-
viding the death rates, and the districts were therefore arranged in ascending
order^ of their size and the presumed errors — 1) were also airanged bj
magnitude vdth -f and - errors alternating, each smoke reading being then
collected by the corresponding factor (F) to estimate the true average for the
district. This led to a correlation coefficient mth lung cancer of 0-8/3, the same
as the original uncorrected value (Table W). The conclusion from these tests is
that the errors arising from the use of only a single instrument site in each
district have not affected the correlation coefficients sufficiently to invalidate the
deductions made from such data in this paper. For greater accuracy it would
be advisable in future stuides of this kind to instal, if practicable, more than one
instrument in districts with population (P) exceeding 50,000. A convenient criterion
for the number (n) to be used would he n = -\/P /20,000, the choice of sites being
related to the population distribution vithin the area as suggested by the Sheffield
study (Clifton et ah, 1959).

Social environment

Several measures of the average social conditions in administrative areas have
been used by statisticians in the study of death rates. The most readly available
one is the density of population per acre, and in international comparisons of
tou'ns this may be the only one which can be obtained from official data. Death
rates from respiratory diseases are highly correlated with this measure of social
conditions, but only in recent years has it been realised that the main reason for
this is the simple fact that population per acre is in itself a measure, in Britain at
least, of the number of domestic chimneys per acre and therefore of the average
density of domestic smoke in the atmosphere of the area. There are other factors
contributing to the correlations, for example districts Arith high densities of
jiojuilation tend to have more heavy industries, more unskilled Avorkers and
])oorer housing conditions. That these other factors are of minor importance
compared Avith smoke in producing the correlation of 0-839 in the 26 northern
areas hetAA'cen lung cancer mortality of males and persons per acre (Table
IS evident from the fact that this figure is reduced to 0-327 when smoke densitA^ is
he d eonstant. Population per acre is not, therefore, a good measure of non-
atinosiilioric social conditions since it is so closely linked vrith smoke densitv that
to liold It constant for 2nd order correlations over-corrects for the social factors

• 11.1 . “ Kina iiaAm been iiwl

■n the proportion of males aged 14 and over in sociaUltssefw and V wS

402

P. STOCKS

employed as a “ social index ” for large tornis (Registrar General, 1936), and that
index has been used here m the study of smoke in county boroughs, and a similar

(fables XI, XIII and XV). Vhere rural areas or small country toums occur in
tJie survey, social class IV may include large numbers of agricultural labourers
wiiose mortality rates are much below the class average, and the proportion in
class V alone is a better social index. For the 26 northern districts this latter
index has been used (Tables III, IV, V, X), and also for the Lancashire and
i orkshire districts but in this case as an alternative the rural districts have been
omitted and the index based on classes IV-V applied (Tables XII and XIV).
In Greater London the correlations between smoke concentration and 4 social
indices were found to be ; persons per acre 0-776, persons per room 0-447, class
IV— V index 0-382, class V index 0-368. In the Lancashire and Yorkshire districts
the coefficient with persons per acre was 0-611, and ivhen rural areas were omitted
the coefficient with class IV-V index was 0-397.

Smoke, polycyclic hydrocarbons and mortality in 26 areas

In Table II the 26 filter locations Avhich Avere chosen ivith a view to measuring
average atmospheric conditions in the districts around tiiem are arranged in order
of the mean smoke concentrations during a complete year, ranging from 15 mg.
per 1000 cubic metres of air in Conway Valley to 562 in Salford. The next 4
columns show the amounts of 3 : 4 benzopyrene, 1 ; 12 benzoperylene, pjT-ene and
fluoranthene in parts per million of the smoke, and the last 4 columns show the
same hydrocarbons in micrograms per 1000 cubic metres of air. The hydrocarbon
rates are based on the improved analytical methods introduced in 1958 and
consequently some are not comparable with rates published previously, but all
figures in the table are comparable one with another.

The geographical variation in amounts of the hj^drocarbons per unit of smoke
is less than that shov'n by the concentrations in air ; for the former the coefficients
of variation of the 4 substances are 35, 39, 37 and 52, and for the latter they are
81, 72, 77 and 71. Irritant effects on the respirator}^ tract might depend on the
number of particles of a particular size and containing a harmful substance, on
the average amount of it present in a particle or simply on the total quantity of
the substance inhaled regardless of its distribution in the particles. Since Avorkers
in very high concentrations of coal combustion products in gas Avorks suffer only
moderate irritant effects, the nature of the particles in domestic coal smoke must
be important, but in comparing effects of ordinal}^ atmospheres polluted by
different amounts of domestic smoke it is to be expected that tJje particles col-
lected by the standard filters aatII be similar in nature and that pathological
effects Avill be most closely related to the total amounts of particular chemical
substances inhaled, that is to say their concentration per unit volume of air
Table II shoAVS that the annual average concentration of 3 : 4 benzopyrene ranged
from 1 yg./lOOO in the purest air to 108 in the most polluted ; for 1 : 12 benzo-
perylene it ranged from 0-4 to 74, for pyrene from 1 to 38 and for fluorant lene

from 3 to 58. j t i a-cod

Table III gives the population densities, social indices and standardisea

mortality ratios for cancer of the lung (males), bronchitis and pneumonia m e
administrative areas where the 26 filters AAmre located. Rates for stomim ^ an
breast cancer are not shown from considerations of space, but the c.i . ■ s

ati^iospheric pollution and jiortality

403

Table II.

Sv,oUaU 4 Pol^to Hyirccarb^s in
26 Lomlities of Northern England and If ales, 195 / or 1 9 .

TI,

Hydrocarbons
(/ig. per g. of smoke)

Hydrocarbons
{fig. per 1000 cu. m. of air)

Location of filter
(ranked according
to smoke density) Y

Conway Valley
Llangefni
Tattcnhall .
Wetherby
Ruthin

Blaenau Ffestiniog
Ripon .

Elland
Flint .

York .

ear

Smoke
(mg./
1000
cu. m.)

105

124

125
150
169
177

Clic.stcr

208

Ormskirk

226

Wrexhnm

264

Lancaster

8 .

276

Darwen

8 .

288

Keigliley

8 .

291

Burnley

8 .

311

Birkenhead .

336

Boolle

362

St. Helens

376

Warrington .

7 *

380

(iateshead

8 .

399

Leeds .

8 .

421

Liverjiool

4.55

(Vrincos Rd.)

Newcaslle.on.

8 .

o05

Tvnc»

Snlfonl

8 .

502

(Regent Rd.)

Mean

260

3:4

Benzo-

pj-rene

198

115

109

108

153

137

123

128

125

109

105

169

152

205

. 123

. 156

100
241

149

189

1 : 12
Benzo-

Py-

Fluoran

perjdene

rene

thene

263

118

169

112

114

105

187

129

121

117

136

123

108

153

1.50

151

105

125

101

182

108

145

115

108

0 109.3

58-

5 94-

* Whamcliffe Street.

other cancer are given.

3 ; 4

1 .- 12

Benzo-

Py-

Fluoran-

pjTene

perj-lene

rene

then©

0-9

0-4

0-8

3-3

6-8

3-1

5-0

4-6

5-1

4-5

4-8

9-2

11-0

7-0

10-0

8-1

9-8

3-4

5-4

5-5

21-0

12-0

20-0

. 15-0

12-0

6-0

10-0

. 15-0

14-0

5-0

7-0

. 25-1

20-8

12-8

15-8

. 24-0

21-0

14-0

20-0

. 19-6

12-6

10-5

13-4

. 26-1

28-0

10-7

16-1

. 24-9

24-7

12-0

15-2

. 20-0

14-0

9-0

14-0

. 36-0

35-0

17-0

25-0

. 32-0

32-0

10-0

18-0

. 32-0

30-0

4-0

8-0

. 47-1

42-3

20-7

23-7

. 51 • 5

49-4

24-9

33-2

. 69-2

52-1

28-0

44-2

. 48-8

41-0

14-3

28-3

. 62-0

50-0

29-0

40-0

. 42-0

40-0

14-0

21-0

. 99-9

74-2

29-8

47-9

. 75.0

73-0

37-0

58-0

. 108-0

62-0

38-0

49-0

. 35-0

29-9

14-6

21-5

ales from lung

cancer

> to be reached.

All the

— V. V..* CIO cv wuuie tiiKea as ana tJiey

range from 23 to 165 for lung cancer, 68 to 122 for other cancer, 18 to 259 for
hroncltitis and 61 to 227 for pneumonia.

A social factor vliich is related indirectly ivith density of population and
dircctlj with lung cancer incidence is the amount of cigarette smoking and it is
not pos.sil)lc to correct for this since complete data are lacking but its ^nnrf
in the Norn, Wafe and Merseyside regifn has been «d\y1

into toliacco .smoking histories of hospital patients. enquiries

heavy sn,„hen< -as f„„,ni7o ^felSrin*" ”

V.ea„ue s„,„he„ .l,e „,bnn,r„^ ^s ahont .J and in mZZZtZ
The effect of actually oorreoting for differences in smoking

404

P. STOCKS

Table 111.— Standardised Mortality Ratios for Cancer, Bronchitis
ill 26 Areas of Northern England and Wales where Smoke
been made.

and Pneumonia
Analyses have

S.M.K.’r in 1950-53*

Administrative
area and countyj

Kant Conway R.D. CA
Llangefni U.D. (1) A

Tarvin R.D. (2) CH
Wotlierby R.D. Y

Ruthin M.B. BE

and R.D.

Ffestiniog U.D. M

Ripon U.D. Y

and R.D.

Elland U.D. Y

Flint M.B. F

York C.B. Y

Chester C.B. CH

Ormskirk U.D. L

Wrexham M.B. DE

Lancaster M.B. L

Darwen U.D, L

Keighley M.B. Y

Burnley C.B. L

Birkeniiead C.B, CH

Bootle C.B. L

St. Helens C-B. L

Warrington C.B. L

Gateshead C.B. DU

Leeds C.B. Y

Liverpool C.B, L

Newcastle-on-Tvne K

Salford C.B. ' L

Notes

f ^ — . A.

Persons

per

Social

Other sites

class

.(V)

index

Lung

cancer

of cancerf

Bronchitis

Peumonia

acre

(M.)

(F.)

(M.)

(F.)

(M.)

ff.)

0-1 .

109 .

181

0-2 .

105 .

110

104

126

100

0-2 .

107 .

0-3 .

94 .

102

101

0-1 .

74 .

0-4 .

01 .

154

143

0-2 .

149 .

146

107

3-2 .

99 .

106

114

123

2-1 .

238 .

112

144

105

118

10-4 ,

102 .

113

106

120

113

11-6 .

150 .

115

1)3

102

1-3 .

142 .

110

109

10-G .

141 .

118

125

O'O

199 .

9.5

108

117

5-2 .

211 .

104

109

112

136

125

2-4 .

lie .

104

119

116

107

lS-1 .

150 .

139

181

134

117

10-6 .

222 .

137

117

103

125

116

180

141

13-1 .

295 .

143

194

164

214

219

13-9 .

227 .

111

102

109

160

141

144

103

18-3 .

240 .

125

118

199

223

132

154

25-7

192 .

113

109

136

111

164

119

13-2 .

135 .

134

104

162

J4I

200

202

28-9 .

245 .

100

119

103

139

138

227

245

20-3 .

100 .

123

118

103

114

113

101

126

34-2 .

209 .

165

122

112

259

240

176

154

(1) With Aetlnvy and Twrcelyn R.D’s. (2) District surrounding Tattenhall. * I950-5t for lung
cancer, f All sites except lung, stomach, breast. J A, Anglesey ; CA, Caeman'on ; CH, Cheshire ;
DE, Denbigh ; DU, Durham ; F, Flint L, Lancashire ; M, Merioneth ; K, Korthurnberland:
Y, Yorkshire West Riding.

liabits between Nortli ^Vales and Liverpool was to reduce the urban/rural ratio
for lung cancer rates in men from 2-4 observed to 2-1, and for smaller towns the
effect 'ivas less than this (Stocks, 1955). A sampling enquiry by the Tobacco
Manufacturers’ Standing Committee (1959) showed that the average daily con-
sumpton of packeted cigarettes per adult in 1956 was 9 in rural districts, 9-6 in
London and about 11 in other large towns. Applying these to the regression
graphs of lung cancer mortality on number of cigarettes smoked in the same kin
of area, the rate in large towns might be enhanced by about 20 per cent because
of this diiference. As a test of the effect this coidd have on the correla ion
between Jung cancer rates and smoke density the S.M.R.’s in Table III vaf®
reduced to that extent for the 8 towns of over 100,000 population and the resulting

atmospheric pollution akd jiortality

405

rebate. apprec-.a..y V — « in

smoking habits.

Table W -CorrMion, in 26 Localities oj Norihem Bn,lnni »»f '

Smofce ani H,dromTbons in Air and Standarduei f {

in Males in 1950 - 54 , Population Density and Social (Class T ) Index.

1 _ _i :t.\. 1

1st order coefficients with

Smoke (total)

3 : 4 Benzopyrene
1:12 Benzoperj'lene
Pyrene
Fluoranthene
Bung cancer

3 ; 4 Benzopyrene
1:12 Benzoperj’lene
Pyrene
Fluoranthene

Lung

cancer

0-873

0-862

0-832

0-751

0-779

0-587

0-464

0-004

-0-180

Persons . _ ,

Smoke per acre index P.P.A. Index

Smoke and hydrocarbons per cubic metre of air

Social

index

2nd order with lung
cancer, with constant

0-927

0-936

0-848

0-876

0-871

0-895

0-856

0-798

0-821

0-839

0-649

0-662

0-667

0-635

0-619

0-735

0-531
0 • 457
0-409
0-250
0-293

0-756

0-738

0-667

0-543

0-622

Smoke

0-288

0-090

0-045

0-020

Hydrocarbons per unit weight of suspended matter

_ 0-604 0-591 . 0-185 0-272

0-411 0-606 . 0-240 0-044

_ 0-073 -0-084 . -0-109 0-096

— -0-248 - 0-252 . 0-052 0-018

Correlations between jjolycyclic hydrocarbons and lung cancer rates

Talile IV shows the correlaton coefficients between lung cancer mortality in
men and the social and air-pollution indices, and partial coefficients when the
social factors are constant. Tor the reasons already stated elimination of persons
per acre reduces the primarjr coefficients unduly, but the figures are given for
comparison with those corrected for proportions of men in social class V which
are free from that objection. Concentrations of the hydrocarbons in air are
highly correlated with lung cancer after eliminating the latter social factor, vath
edds exceeding 1000 to 1 against such coefficients arising fortuitously in the case
of 3 of them. All coefficients exceeding 0-375 are significant at the conventional
level of 1 in 20 probability. Amounts of the substances per unit of smoke show
no significant associations with lung cancer, and only in the case of 3 : 4 benzo-
])yrcnc is there any suggestion that this may be of any importance.

Table VI indicates in its first section that the hydrocarbon concentrations in
air are ven,' highly correlated one with another, owing to their common on’o-in
and when a substance x is present in large amount the others must tend to be
nwreased also m consequence. If ar is concerned in disease incidence and the
others arc not, the correlations between the others and the disease rates should
become mappreciahle when the concentration of a; is held constant. In effect this
meaas that m 2G areas having the same degree of air pollution by the active
snhstanco x no correlations would be found between lung cancer mortalitv anrl
amounts of the otiicr hydrocarbons present in the ok In X

-Hu-oml seffiion of the table the concUation of ea J hydr^ttn Tas be" L n

constant m turn, with the following result for lung cLcer Wth 3 - ^

H, total smoke coefficient is reduL from wT.“o

406

P. STOCKS

0J89, indicating the probable presence of some other constituents of smoke which
aiiect the lung, such as certain trace elements as will be seen later ; the benzo-
perjdene coefficient is reduced to 0-055, and the other two hydrocarbons cease to
show any relation witli lung cancer. With 1 : 12 benzoperjdene held constant the
3 : 4 benzopyrene coefficient is still significant (0-394) Avhereas the others dis-
appear ; and the making of p 3 wene and fluoranthene constant scarcely affects the
benzopjTene coefficient and leaves the benzoperylene figure still significant. As
the figures at the foot of the first column indicate, pyrene and fluoranthene
derive their statistical connections with lung cancer from their association with
3 : 4 benzopjT-ene in smoke ; 1 : 12 benzoperylene may have an independent
elfect, but if so it is small in comparison AAoth that of 3 : 4 benzopjwene which
clearlj'^ emerges from this analysis as the hydrocarbon of outstanding importance.
TJiese results agree noth e.xperimental evidence about the carcinogenic potency of
the hydrocarbons.

Correlations behveen polycyclic hydrocarbons and mortality from other diseases

Table V shows no appreciable correlations between amounts of smoke or any
of the lij’^drocarbons in air and mortality from cancer of the stomach in either
sex or cancer of the breast or other organs in females after correcting for social
index. Other cancer in males, for Avhich figures are not sho-nm in the tables,
gives primary coefficients of 0-64 with smoke, 3 : 4 benzopyrene and pyrene,
0-63 AA'ith 1 ; 12 benzoperylene and 0-61 noth fluoranthene, and when social index
and other hydrocarbons are constant the benzopyrene and pyrene coefficients are
still appreciable but not significant and no conclusion can be drawn. The group
includes the larynx, oesophagus and intestine, of which separate data are not
obtainable.

Bronchitis gives primary correlations similar to those for lung cancer, and the
partial coefficients when social index is constant are significant for males in
respect of each hj^drocarbon and for females in respect of 3 : 4 benzopjwene and

Table V . — Correlations in 26 Localities of Northern England and Wales between
Smoke and Hydrocarbons in Air, Standardised Mortality from Cancer of the
Stomach, Breast and Other Sites, Bronchitis and Pneumonia in 1950-53, and
Social {Class V) Index.

Smoke (total)

3 : 4 Benzopyrene
1:12 Benzoperylene
Pyrene
Fluoranthene

Cancer (excluding lung)

A — ,

Stomach Bronchitis Pneumonia

^ Ri-rfinct. Othpr 1

' (M.)

k ^

(F.)

Breast

(F.)

Other

(F-)

r“

(M.)

(F.) '

' (M.)

(F.)'

1st order

coefficients

0-241

0-308

0-104

-0-002

. 0-869

0-751

. 0-666

0-479

0-261

0-305

0-136

0-024

. 0-809

0-099

. 0-699

0*549

0- 141

0-281

0-129

0-013

. 0-741

0-672

0-715

0* 511

0-045

0-219

0-071

0-093

. 0-683

0-530

0-662

0*443

0-118

0-319

-0-022

0-101

. 0-700

0-451

0-636

0*583

Smoke (total) . . 0-123

3 : 4 Benzopyrene . 0-148

I : 12 Benzoperj’leno . Nil
PjT-ene . . .

Fluoranthene . .

2nd order coefficients n-ii

0-077 — —

0-073 — —

0-048 — —

0-077 — —

0-140 — —

social index constant

0-768

0-570

. 0-491

0-256

0-693

0-513

. 0-540

0-350

0-532

0-430

, 0-505

0-297

0-448

0-211

. 0-490

0-209

0-489

0-094

. 0*455

0-418

atmospheric pollution and mortality

407

1 : 12 benxope^lene. TaWe

held constant the other hydrocarbons c ^ ^ average 0-584 when the others
with bronchitis mortality significant correlations when social index is

“pr:t

Jnter-correlalions between Smoke and Hydrocarbons

Benzo-

Benzopyrene

3 ; 4 Benzopyrene .
1:12 Benzoperj'lene
Pyrene .
Fluoranthene .

Smoke

0-927

0-93C

0-848

0-876

0-956

0-915

0-917

peiylene

0-956

0-902

0-927

Pyrene

0-915

0-902

0-951

Fluoran-

thene

0-917

0-927

0-951

Benzopyrene constant
Smoke (total)

1; 12 Benzoperj'lenc

Pyrene

Fluoranthene

Benzoperylene constant
Smoke (total)

3 ! 4 Benzopyrene

Pyrene

Fluoranthene

Pyrene constant
Smoko (total)

3 : 4 Benzopyrene
1:12 Benzojierj-lene
Fluoranthene

p'luoranthcne constant
Smoke (total)

3 -. 4 Benzopyrene
I ; 12 Bcnzopcrj-lenc
Pyrene

Correlations

with Indices of MortaUty

Cancer of

Bronchitis

Pneunr\oniR
A

mi "(Md (M.) (F.)

' 2nd order coefficients with one hydrocarbon constant

0-389

0-538

0-386

0-068

Nil

0-055

Nil

♦ >

Nil

• »♦

• if

0-017

xVil

0-223

0-081

0-240

0-480

0-741

0-470

0-073

0-002

0-394

0-511

0-259

0-237

0-002

0-040

Hit

0-058

Nil

0-065

0-077

0-333

0-674

0-748

0-671

0-262

0-173

0-654

0-625

0-624

0-306

0-443

0-542

0-397

0-535

0-363

0-288

0-318

0-225

Nil

0-026

0-581

0-629

0-741

0-828

0-293

0-589

0-586

0-899

0-376

0-043

0-408

0-344

0-760

0-434

Nil

0-055

0-058

0-366

0-242

Per cent reduction of smoke coefficients by
eliminating one hydrocarbon

Jlydroearbon constant

3 : 4 Bfn/opyrcnc

100

1 : 12 Uonzoperylenc

100

IVrom^

Kiuorautlioiie

100

Ilrnznpt/rrnr constant
1:12 Bcnroperylene
Pyrene
Pliinrantbeiie

Per cent reduction of other hydrocarbon correlations
by eliminating 3 : 4 benzopyrene

100

69 100

88 100

100 59

408

P, STOCKS

fluoranthene, but in Table Yl tlie results of eliminating the substances individually
are different from those for lung cancer and bronchitis. When 1 ; 12 benzo-
perylene is made constant all the other correlations disappear for males whilst
fluoranthene remains predominant for females, and this suggests that these may
be concerned with the incidence of some cases of pneumonia whereas benzo-
pyrene is less important.

Trace elements and mortality in 23 areas

The amounts of 13 elements in the smoke samples were determined spectro-
graphicalty at the Fuel Research Station of the Department of Scientific and
Industrial Research by JWr. K. V. Aubrey. Filter papers were changed at intervals
of a week or longer and the suspended matter collected throughout a whole year
was aggregated for analj^sis. In 1956 when 10 stations were operating about
500 cubic feet of air per week passed through a standard 2-inch or a special 4-inch
diameter filter according to the degree of air-pollution anticipated ; but in 1957
and 195S when more stations were introduced about 3000 cubic feet per week
were drawn through a 4-inch filter to obtain larger quantities of smoke for analysis.
This was done at 23 of the 26 representative locations in Table I, and the results
are given in Tables Vila and VII6. Analyses of copper and chromium are
available for 14 of these.

Table Vila. — Average Annual Amounts of Total Ash, Lead, Zinc, Copper, Tita-
nium, Arsenic and Manganese per 1000 cubic metres of Air at 23 Localities in
Northern England and Wales in 1956-58.

Locality

Conway Valley .
Llangefni .

Tattenholl .

Wetherby .

Kuthin

Ripon

Elland

Flint ....
York . . . ■

Chester
Ormskirk .

Wrexham .

Lancaster .

Darwen

Keighley

Burnley

Birkenhead

Bootle

St. Helens .

Warrington

Leeds

Liverpool (Princes Rd.)
Salford (Regent Rd.) .

mg.

r -t

Year

Total

ash

Lead

1957

4-0

0-08

1950

0-27

1950

8-0

0-22

1958

6-6

0-35

1957

8-5

0-47

1958

6-5

0-40

1958

7-2

0-42

1950

12-5

0-29

1958

11-4

0-66

1950

12-0

0-50

1957

10-0

0-52

1950

10-0

0-39

1958

8-9

0-63

1958

11-6

0-71

1958

13-0

0-71

1958

10-1

0-95

1956

13-0

0-60

1956

22-0

0-58

1956

15-0

0-44

1957

24-0

0-75

1958

37-1

1-30

1950

15-0

0-57

1958

21-0

1-43

;Cole . — The localities are ranked in order of smoke

Man-

Zinc

Copper

Titanium Arsenic

ganese

ISO

—

205

100

285

230

—

295

250

225

250

—

185

140

185

240

490

330

—

130

160

440

370

180

130

280

335

252

■io

density as in Table II.

ATMOSPHERIC POLLUTION- AND MORTALITY

409

Table VII6 . — Average Annual Amounts of Nickel, Antimony, Vanadium, Chro-
mium, Molybdenum, Cobalt and Beryllium per 1000 cubic metres of Air at 23
Localities in Northern England and Wales in 1956-58.

/‘g-

Locality

Conway Valley
Llangefni
Tattenhall
Wetherby
Ruthin

Bipon
Elland
Flint .

Vork .

Chester
Ormskirk
Wrexham
Lancaster
Dnnven

Keighley
Burnley
Birkenhead
Bootle
St. Helens

U'arrington
Leeds

Liverpool (Princes Rd
•Salford (Regent Rd.)

Year

Nickel

Anti-

mony

Vana-

dium

Chro-

mium

Molyb-

denum

Cobalt

Beryl

Hum

1957

1-3

0-0

1-2

0-9

0-29

0-25

0-00

1956

2-3

4-0

3-4

—

0-20

0-70

0-20

1956

4-1

5-3

6-9

—

0-40

1-30

0-20

1958

74* 5

5-3

1-6

2-7

1-30

0-85

0-14

1957

1-8

3-3

1-2

1-6

0-60

0-74

0-1.5

1958

31-2

4- 5

1-5

-2-9

1-19

0-83

0-14

1958

205-0

8-6

1-1

2-1

1-25

2-30

0-19

1956

6-9

7-0

5-9

—

1-00

1-20

0-.50

1958

29-0

7-7

5- 1

5-4

2-35

1-13

0-27

1056

12-0

7-6

1-90

1-60

0-60

1057

16-0

12-5

4-6

2-7

1-40

0-97

0-26

1956

6-9

8-8

4-6

—

1-40

1-30

0-60

1958

17-3

7-2

5-4

2-3

1-56

0-78

0-21

1958

11-2

9-5

8-8

4-0

3-40

1-05

0-31

1958

32-5

13-0

3-7

4-1

2-50

2-05

0-31

1958

13-5

8-6

6-8

4-8

4-45

1-55

0-40

1956

10-0

12-0

31-0

—

2-00

1-60

0-60

1950

23-0

20-0

42-0

1-80

2-00

1-00

1956

24-0

250-0

18-0

—

3-20

1-60

0-90

1957

37-0

21-5

12-0

4-4

5-00

2-90

0-81

1958

23-0

16-0

15-0

21-5

6-10

4-25

0-84

1956

11-0

16-0

30-0

2-70

1-40

1-00

1958

32-0

18-0

14-2

7-0

6-60

2-60

0-67

are ranked

in order of smoke density

as in Table II.

For a few of the elements one or two places showed concentrations outside

: wT^s^eifoi ti,"e

cent. The concentrations of 11 elements were ^

.iiortality ratios in Table III with results shoyvn in Tabl^ vSl-X.® standardized

ociitr.nion in nir is coiTclatcd°BigSfi|ja*I!'SbT"’'’* “on-

f, ' i" of siBnifiS„« S"?!' '""I mortahV, the critical

bp loisl .sniokc coeliicicnt (0-SG0\ of makinw tb. ^ahle IX reveals the effect on
■OKI imly for 11,0 first .i elemeMs herl!S? constant

;::'v = s, nit, r t

410

P. STOCKS

Table Vlll.—Comlations in 23 Localities of Northern Enqland and Wnh, h.,,..

0 / Trace mcnenUr .» Air, SlanJardiscd MomSi fror^cTmr
Broncjnks and Pneunmma in 1950-53 and Proportion of aqed U and
ovei tn Social Class V in the Population.

Beryllium
Arsenic .

Zinc

Jlolyfadonum
Vanadium
Cobalt .
Nickel .
Manganese
Lead
Titanium
Antimony

Social index .

Beryllium
Arsenic .

Zinc

Molybdenum
Vanadium
Cobalt .
Nickel .
Manganese
Lead
Titanium
Antimony

Social index .

1st order coefficients

Cancer of -

Other

cancer

( —

except stomach

Lung

Breast

A ,

(M.)

(F-)

(M.)

(F.)

0-827

0-129

0-550

0-124

0-748

-0-080

0-321

0-086

0-759

0-165

0-405

0-054

0-G84

0-028

0-520

0-109

0-770

0-053

0-556

-0-061

0-536

0-220

0-296

0-063

-0-095

0-112

-0-24]

-0-110

0-521

0-351

0-400

0-162

0-626

-0-017

0-462

0-149

0-527

0-082

0-338

0-106

0-327

0-132

0-230

0-148

0-724

0-149

0-452

0-266

Bronchitis

Pneumonia

' (M.)

(F.)

' (M.)

(P.)

0-767

0-642

0-760

0-783

0-743

0-513

0-684

0-643

0-796

0-547

0-615

0-674

0-842

0-816

0-482

0-447

0-620

0-463

• 0-805

0-711

0-710

0-599

0-498

0-497

0-095

-0-015

-0-053

0-020

0-556

0-473

0-310

0190

0-778

0-722

0-489

0-334

0-602

0-597

0-495

0-512

0-380

0-239

0 132

0-333

0-049

0-598

0-639

0-447

Ash

(total)

0-729

0-334

0-765

0-746

0-513

0-692

0-022

0-928

0-90S

0-936

0-261

0-452

Social

index

0-670

0-745

0-676

0-399

0-763

0-215

-0-232

0-289

0-315

0-223

0-211

coefficients exceed 0-53 and tlie arsenic and zinc coefficients remain appreciable
though not significant at the conventional level. Both berjdlium and molyb-
denum appear to be associated with lung cancer incidence, and a suitabty weighted
combination of the ttvo elements produces a high correlation of 0-720 with mortality
after eliminating social index.

The connection with beryllium is supported by some experimental evidence
of bronchogenic cancers in rats after inhaling berj>^llium dust for a long period, and
several cases of lung cancer in men following berylliosis have been reported.
Beryllium dust is known to be a respiratory irritant and many hundreds of cases
of clmonic berylliosis have occurred in processing the metal fi:om ores (Hueper,
1957). It is of considerable interest to now find epidemiological indications of a
connection betrveen the small amounts of this element in ordinory air and the
incidence of lung cancer amongst men who are continually inhaling it. Experi-
mental evidence for any carcinogenic elfects of molybdenum seems to be lacking,
but this may’’ be because it has not been sought for ; the very^ high correlations
noted below with bronchitis in both sexes are remai'kable and the element tnig
well be concerned also in lung cancer incidence.

ATMOSPHERIC POLLUTION AND MORTALITY

411

Arsenic is correlated significantly (0-427) with lung cancer after eliminating
the beryllium and motybdenum, and this tends to strengthen a view, long held,
that this element in oxide form can have carcinogenic effects on the lung. Zinc
gives a coefficient not quite significant at 1 in 20 level (0-379) and this might be
meaningful in view of the evidence for a connection between its concentration in
soil and the incidence of cancer of the stomach (Stocks and Davies, 1960). Vana-
dium also retains an appreciable correlation (0-347) after eliminating beryllium
and molybdenum, and since it is a respiratory irritant some attention might be
paid to it along Avith molybdenum as possible carcinogenic agents despite the
present absence of clinical or experimental evidence.

Correlations between trace elements and other diseases

Table VIII shows no significant correlations between mortality from breast
cancer and concentration of any trace element. The coefficient with manganese
is reduced to 0-325 when social index is constant. Other cancer (excluding
stomach) in females likewise shows no appreciable association Avith amount of
smoke or of any element. In males however there is a smoke correlation of
0-603, Avhich is reduced considerably by separate elimination of beryllium, molyb-
denum and vanadium. When social index and molybdenum concentration are

Table IX. — Correlations between Mortality and Amount of Smoke in Air of 23
Localities when the Effect of one Trace Element on the Coefficient is Eliminated.

Smoke

Lung

cancer

(M.)

0-860

Other Bronchitis

cancer* . , '

(M.) (M.) (P.)

1st order coefficients with smoke
0-603 . 0-919 0-843 .

Pneumonia

I A

(M.) (F.)

0-712 0-564

Connlant factor

Beryllium
Arsenic .

Zinc

Jlolybdenum
Vanadium
Cobalt .
Nickel .
Manganese
Lead
Titanium
Antimony

2nd order coefficients with smoke, one element constant

0-479 . 0-321

0-712 . 0-570

0-733 0-489

0-740 . 0-360

0-767 . 0-307

0-811 . 0-692

0-880 . 0-602

0-815 . 0-498

0-872 . 0-474

0-812 . 0-535

- 0-842 . 0-573

0-791

0-822

0-847

0-735

0-846

0-888

0-936

0-881

0-787

0-871

0-905

0-498

0-801

0-768

0-521

0-785

0-752

0-848

0-793

0-630

0-753

0-838

0-242 Nil
0-425 0-179
0-504 0-189
0-634 0-385
0-430 0-273
0-592 0-377
0-711 0-568
0-685 0-552
0-686 0-632
0-609 0-366
0-723 0-501

Beryllium
An^enio .

Zinc

Molybdenum
Vnmulium
Cobnlt .
Xiobol .
Mnnpuip^e

Titanium

Aiitimonv

•17

. 5

* Excluding st

Per cent reduep’on of smoke coefficients bv
eliminating one trace element

3
0

4
14

5
I

5
9

6
25
11

100

32
55

33
0
2
0

412

P. STOCKS

held constant the correlations of other cancer with zinc, manganese and lead
disappear bnt the coefficient with vanadium remains significant (0-396). This
suggests that some forms of male cancer as well as lung may be affected by berjd-
lium, molybdenum and vanadium.

Bronchitis shows in Table VIII correlations similar to those of lung cancer,
and in tiie case of females the high coefficient noth smoke is seen from Table IX
to be reduced considerably by eliminating beryllium, molybdenum and lead.
When social index and molybdenum are held constant the correlations with
beiyllium and lead virtually disappear, but molybdenum retains a high coeffi- •
dent of 0-706 with female bronchitis after eliminating social index and berjdiium.
Bronchitis in males shows relations noth the elements similar to those of lung
cancer. IVith social index and ber 3 fltium constant the correlation with molyb-
denum is 0-712 ; when social index and molybdenum are ehminated arsenic and
vanadium give coefficients of 0-563 and 0-469, beryllium gives 0-366 and zinc
0-360 ; and wlien social index and arsenic are constant molybdenum gives 0-S50,
berjdb'um 0-360 and zinc 0-425. It appears that molybdenum in the air is eon-

Table X. — Correlations in 23 Localities between Concentrations of Trace Elements
in Air and Mortality when the Social Index and Other Trace Ele7ne7iis are
Held Constant.

2nd order inter-correlations between trace elements
wlien social index is constant

Molyb-

denum

Arsenic

Vana-

Zinc dium Cobalt

Man-

ganese

Lead

Beryllium
molybdenum .

Both together
l'’anadium

Zinc

. 0-495

.’ m

. 0-756

. 0-293

. 0-224

. 0-496

. 0-541

. 0-652 . 0-610 . 0-552 .

. 0-492 . Nil . 0-771 .

. 0-406 . 0-259 . 0-719 .

_ . — . 0-1G8 .

0-670 ’.

0-311

0-849

3rd order coefficients with lung cancer, eliminating
social index and one or more trace elements

Element constant

Berj-llium
Molybdenum
Both together
Arsenic

Beryl-

lium

, 0-552

! 0-587

Molyb-

denum

. 0-451

.' 0-591

Vana-

Arsenic Zinc dium

! 0-389 . 0-337 . 0-538 .

. 0-427 . 0-379 . 0-347 .

Cobalt

0-307 .

0-096 .

Lead

Similar coefficients with other diseases
Bronchitis (males)

Beryllium

Molybdenum

Arsenic

! 0-366

. 0-360

. 0-712

! 0-850

! 0-563 . 0-360 . .0-469 .

_ . 0-425 . —

0-296 .

0-294

Bronchitis (females)

Beryllium

Molybdenum

! 0-046

. 0-706

0-144

Beryllium
Vanadium .

.’ 0-316

. 0-048

. 0-416

Pneumonia (males)

Nil . Bil . 0-443 .

! 0-136 . — • — •

0-236 .

—

Beryllium

Nil

Pneumonia (females)

Nil . 0-174 . Bil

0-2C8 .

—

413

atmospheric pollution and JIORTALITy

nected with bronchitis mortality in both sexes, and that
bervlhum and zinc may be concerned also m the case of males. The effects of
beryllium on the respirator}^ tract have been referred to m reference to lung
cancer. Vanadium pentoxide dust has for long been knovm to be an irritant to
the upper respiratory ' tract, sometimes producing bronchitis or pneumonia
amongst men working with it (Wyers, 1946; Sjoberg, 1950, 1956).

Pneumonia shows strongest primary correlations Avitli beryllium, vanadium,
arsenic and zinc, and the coefficients wdth smoke, which are not so great as for
bronchitis and lung cancer, are reduced considerably by eliminating any of these
elements. When social index and beryllium are held constant a significant
coefficient remains only for vanadium {0-443) in the case of males, and all cor-
relations disappear or become insignificant for females. It appears that beryllium
is important for pneumonia in both sexes, and vanadium also in males.

Association of standardised mortality from cancer, bronchitis and pneumonia with
amounts of smoke and atmospheric deposit in urban areas of England and
Wales

In a pre^^ous paper (Stocks, 1959) correlations for cancer and bronchitis were
given in respect of smoke collected by filters and undissolved deposit of larger
particles in the county boroughs of England and Wales and districts of Lancashire
and the West Riding of Yorkshire where data were available for one or more
years from the reports of the D.S.I.R. The present study extends that analysis
in the following ways •. (1) records have become available for some new areas
(2) cancer of the lung has been examined for each sex and cancer of the oesophagus
distinguished where possible, (3) pneumonia has been added, (4) a similar study
has been made of all the parts of Greater London for which data are available,
(5) social conditions have been assessed by proportions of men in unskilled and
partly skilled occupations. In towns where measurements had been recorded at
multiple sites the mean value, after excluding instruments sited to investigate
specific sources of pollution, was taken as the index of pollution for the tonm.
The expected deaths for the standardised mortality ratios in 1950-53 were cal-
culated by multiplying the England and Wales death rates at 12 sex-age groups
in those years by the corresponding local populations in the 165 areas.

Almosphenc deposit (undtssolved).— Table XI shows the correlations with the
average amount of such deposit falling on unit area of the deposit gauee ner
month in 53 county boroughs. Coefficients exceeding 0-266 are significant at
1 m 20 probability level, those exceeding 0-345 at 1 in 100 level and thos^e exceeding
0-4.1/ at 1 m 1000 evel. The coefficients with lung cancer, bronchitis and

the West

iho huier gro„|, coefficients exceeding 0-230 are significant

rnt h... eanccc and igonei.itis, i,nt gh^nt

414

P. STOCKS

organs other tlian tJie lung show no appreciable correlations when social index is
constant, but stomach cancer in females gives a significant coefficient in the 74
districts when persons per acre are made constant.

Table XI. — Correlations in 53 County Boroughs between Amounts of Atmospheric
Deposit (Undissolved) and Standardised Mortality from Cancer, Bronchitis and
Pneumonia in 1950-53, Persons per Acre and Proportion of Males Aged 14
and Over in Social Classes IV-V in 1931.

2nd order

1st order coefficients

with deposit

(constant idices)

Persons

Social

Sex

Deposit

per acre

index

P.P.A.

Social

Cancer of

Lung (1950-34)

. U. & F. .

0-536

0-650

0-391

. 0-531

0-477

Oesopliogus ,

. M. .

0-114

0-192

0-228

0-009

0-061

-0-193

-0-103

-0-133

. -0-176

-0-I7I

Stomach

. M. .

0-414

0-077

0-3G0

0-408

0-344

F. .

0-383

0-111

0-401

. 0-374

0-301

Intestine

. M. & F. .

0-179

0-219

0-143

0-175

0-144

Breast .

. F.

-0-171

-0-075

-0-337

. -0-158

-0-078

Other sites

. M. .

0-224

0-419

0-028

. 0-004

0-226

F. .

0-097

0-126

-0-025

. 0-072

0-109

Bronchitis

. M. .

0-631

0-346

0-504

0-579

0-570

F. .

0-540

0-245

0-379

0-511

0-483

Pneumonia

. M. .

0-503

—

0-404

. —

0-431

F. ,

0-685

—

0-487

- —

0-647

Persons per ere .

0-219

. —

—

Social index

. —

0-300

—

. —

—

Table XII. — Correlations in 74 Administrative Areas of Lancashire and the (Vest
Riding of Yorkshire betiveen Amounts of Atmospheric Deposit (Vndissolved),
Standardised Mortality from Cancer, Bronchitis and Pneumonia in^ 1950-53,
Persons per Acre and Proportion of Males Aged 15 and Over in Social Classes
IV-F in 1951.

Sex

Cancer of

Lung (1950-54)

. M. & :

Stomach

Breast

. F.

Other sites .

. M.

Bronchitis

. M.

Pneumonia

. M.

Persons per acre

. —

Social index IV— V .

—

All areas with deposit data

I ~ '

]st order, irith 2nd

, 1 , order.

Deposit

Persons

per

acre

Deposit

{P.P.A.

constant)

0-353

0-611

0-260

0-221

0-140

0-189

0-226

0-014

0-232

0-041

-0-017

0-046

0-258

0-313

0-187

0-114

0-099

0-482

0-458

0-407

0-541

0-334

0-490

0-282

—

0-305

—

— ■

0-305

—

Excluding Bural Districts

Jst order

^ - -

2nd

order.

Deposit

(index

— >

Social

index

Deposit

IV-V

constant)

0-358

0-125

0-400

0-206

0-365

0-030

0-329

0-399

0-158

0-038 -

-0-201

—

0-237

0-310

0-102

0-068

0-254

_0-070

0-452

0-540

0-340

0-588

0-420

0-482

0-303

0-346

0-150

0-370

0-399

0-216

—

0-498

—

atmospheric pollution and mortality

415

Atmospheric particles large ” VXt

unlikely to reach the tog, ^ smoke filters, and this ma}' account

excess of the finer particles v.h P conditions With stomach cancer there

p“Sa fan ppop unprotected food and soil

the hands, being then ingested.

tr YTTT rnyrflaiions in 30 Couniv Boroughs between Amount of Smoke in

fr^Jcanur. ZZToZ Z

1950-53 Persons per Acre and Proportton of Males Aged 14 and Over

Social Classes IV-V in the Population in 1951.

2nd order

Cancer of

Lung (1950-34)

Oesophagus

Stomach
Intestine
Other sites

BroncliilU

Pneumonia

Persons per acre
Social index IV-V

1st order coefficients

with smoke

(constant indice.s)

Persons

Social

per

index

Clas.s

Sex

Smoke

acre

IV-V

p.p.a.

IV-V

0-494

0-448

—

0-326

0-491

0-468

. —

0-309

M. & F.

0-5-20

0-480

. 0-451

0-339

0-361

0-220

0-368

. 0-319

0-202

0-016

0-014

—

, 0-013

—

M. & F.

0-685

—

0-.539

—

0-546

M. & F.

0-366

—

0-041

. —

0-466

0-164

0-313

0-227

. 0-078

0-044

0-021

—

-0-160

. —

0-042

0-604

0-313

0-587

. 0-364

0-405

O-SIO

0-380

0-499

. 0-325

0-410

0-493

0-409

0-563

. 0-426

0-240

0-332

0-153

0-539

. 0-303

0-036

. ■

0-297

0-569

—

Smoke . — Table XIII shows the correlations with average annual amounts of
smoke per unit volume of air in 30 county boroughs where filters had been operat-
ing. Coefficients exceeding 0-349 are significant at 1 in 20 level of probability,
those exceeding 0-449 at 1 in 100 level and those exceeding 0-554 at 1 in 1000
level. The coefficient with lung cancer when population density is constant is
(1-4')! and when social index is constant it is 0-339. Bronchitis and cancers of
the stomach and intestine also give significant correlations after eliminating the
social factors, hut the pneumonia figures are not so definite. Cancer of the
oe-sophagus gives coefficients of doubtful significance and cancer of other sites
siiows no associations with smoke.

Table Xll' give.s coefficients in 45 districts of Lancashire and the West Ridino-
where filters had been operating, and in 42 after excluding rural districts, those
significant at ; in 20 probabilitj^ level and those exceeding

o-.L-at 1 m lOOlevel. The correlations inth lung cancer remain highly significant

416

P. STOCKS

Table XI’\ .—Correla(to7is m 45 Administrative Areas of Lancashire and the West
Riding of 1 ortshire between Amounts of Smoke in Air, Standardised Mortaliin
frorri Cancer Bronchths and Pneumonia in 1950-53, and Proportions of Mali
in Social Classes IV~V in 1951. ■'

Sox

Cancer of

Lung (1950-54)

. JI. &

Stomach

. M.

Breast

. F.

Other sites .

. lU.

Bronchitis

. M.

Pneumonia

. JI.

Socinl class IV-V . —

indices V . —

All areas with smoke data

1st order

2nd

order.

Smoke

(index

CIoss

Smoke

index

constant)

0-558

0-577

0-353

0-200

0-050

0-288

0-230

-0017

0-310

-0-002

-0- 160

0-125

0-343

0-342

0-181

0-034

-0-037

0-073

0-440

0-389

0-280

0-320

0-258

0-270

0-442

0-453

0-232

0-423

0-314

0-303

0-301

0-019

—

Excluding Rural Districts
1st order 2nd

{

■

oraer.

Class

Smoke

IV and V

(index

Smoke

index

constant)

0-531

0-149

0-519

0-230

0-385

0-091

0-225

0-483

0-041

-O-OOG

-0-319

0-139

0-319

-0-026

0-390

-0-001

0-068 -

-0-030

0-435

0-443

0-315

0-307

0-253

0-232

0-406

0-458

0-274

0-426

0-460

0-299

0-397

0-689

—

Table XV . — Correlations in 40 Areas of Greater London behveen Amount of Smoke
in Air, Standardised Mortality in 1950-53 from Cancer, Bronchitis, and
Pneumonia, and Proportion of Males Aged 15 and Over in Social Classes
IV and V in the Population.

1st order coefficients

' , 2nd order with smoke

Socinl indices (constant indices)

Sex

Smoke

t ^

IV-V

V only

IV-V

-A ^

V onh

Cancer of

Lung

. U. .

0-408

0-684

0-695

. 0-221

0-228

F. .

0-386

0-368

0'37}

0-284

0-292

Stomach

M. .

0-330

0-809

—

0-032

—

F. .

0-203

0-579

—

. -0-025

—

All other sites

. M. .

0-164

0-514

. -0-042

—

0-325

0-278

> — ■

0-246

—

Bronchitis

. M. .

0-454

0-773

—

0-269

—

0-566

0-698

• —

0-299

—

Pneumonia

. M. .

0-530

0-695

- —

0-410

—

0-466

0-584

' —

0-322

—

Social class IV-V

0-384

—

indices V

—

0-368

—

- —

—

Table XV shows the correlations in 40 areas of Greater London where smoke
data were available, the number comprising 15 districts in the Outer Ring, 24
Metropolitan Boroughs and a central area covering Holburn, CitjL Finsburj' and
Shoreditch. In view of the great daily movement of people, particularly of men,
from one district to another it would hardly be surprising to find little or no
correlation between mortality of residents and the average smoke densitj' in le
area of residence when social differences have been allowed for. Coefticien s
exceeding 0-304 are significant at 1 in 20 level, and the lung cancer and broncliitis

ATMOSPHEKIC POLLUTION AND MORTALITY'

417

correlations are slightly belo^Y this level, though the pneumonia figures are
significant for males and females. Stomach cancer shows no associations with
sLke nor does cancer of other sites in males, but for other sites m females there
is a small but not significant correlation. The Greater London results are in
general accord with those from the other groups of areas, though as was expected
the associations with smoke are weaker.

STTMaiARY'

Lung cancer mortahty is strongly correlated with smoke density in the
atmosphere in 26 areas of Northern England and Wales, in 45 districts of Lanca-
shire and the West Riding of Yorkshire, and in 30 county boroughs, whilst similar
though weaker correlations are found within Greater London. These relations are
only partially explicable by social differences in the populations concerned.
Bronchitis and pneumonia in males and bronchitis also in females show similar
strong correlations with smoke. Cancers of the stomach and intestine are related
significantly with smoke in the county boroughs, and cancers of sites other than
lung and stomach in males are so related in the other groups of areas. In females
cancers of the breast and other organs show no association with smoke. Pollution
by larger particles is related significantly ndth lung and stomach cancers, bronchitis
and pneumonia in 53 county boroughs and Yvith lung cancer and bronchitis in 74
districts of Lancashire and the West Riding.

In the 26 localities the smoke samples were analysed in respect of polycyclic
hydrocarbons and a statistical process of successive elimination was applied to
discover which hydrocarbon was responsible for the smoke correlation ivith
mortality rates. Por lung cancer and bronchitis 3 : 4 benzopjTene emerges
clearly as the substance of prime importance, with 1 : 12 benzoperylene contri-
buting weakly for lung cancer, but for pneumonia 3 : 4 benzopyrene is apparently
not important. The composite group of other cancers in males is correlated with
several hydrocarbons, but cancers of the breast and other sites in females show' no
relations with any of them.

In 23 localities spectrographic analyses for 13 trace elements w'ere made and
a similar process of successive elimination was applied to those w'hich showed
api)reciable correlations with mortality rates. Por lung cancer benilium and
molyhdenmn emerge as the elements of most consequence, with arsenic zinc and
vanadium showing weaker associations. Por bronchitis molybdenum appears to
Ijc the ...tportant clement in both sexes n hilst in males beo-Uium, a”eS ™„a-
tlrnm and ,.mc may also be concerned as for tang cancer. Por pnenmonia
hum emerges as the important element in both sexes with vanafwTcrf ^
cernocl in males. With other cancer in males

vanadium .show associations but breast and nf-har- ^ and

relations with any elemenr show no

eontrilmtcd to the early strgrs^S^hjJco^OTOrath'r^^^ people w'ho

larly to Mr. R. T. Commins of the Medical ^ indebted particu-

Laboratory for tlie analvtical work on the hvdroop 1 Group at the Dunn

llw Fuel He,earcl. Station of

crcarcl, for tbe .spectrograpbie tvork, and have te thanS R.

418

P. STOCKS

Dr. B. T. Wilkins of those departments for help and advice, the General Register
Office and County Medical Officers for supplying mortality data and the Health
Officers of all the districts where instruments were installed for their co-operation.

This wmrk was supported by grants from the British Empire Cancer Campaign
and Medical Research Council.

REFERENCES

Clifton, M., Kerridge, D., Moulds, W., Pemberton, J. and Donoghue, J. K.—
(1959) hit. J. Air Pollution, 2, 188.

CoMMiNS, B. T. — (1958) Analyst, 83, 386.

Hueper, W. C. — (1957) ‘ Cancer ’, Vol. 1. London (Butterworth).

Registrar Generad. — (1936) Statistical Review for 1934, Text.

Sjoberg, S. G. — (1950) Acta med. scand. (Suppl. 238), 138, 1. — (1956) Ibid., 154, 381.
Stocks, P. — (1955) J. Fac. Radiol., Lend., 6, 166. — (1958a) Supplement to 35th Rep.
Frit. Emp. Cancer Campgn. — (19586) hit. J. Air Pollution, 1, 1. — (1959) Brit. med.
J., i, 74.

Idem and Campbell, J. M. — (1955) Ibid., ii, 923.

Idem, Commies, B. T. and Aubrey, K. V. — (1960) Ini. J. Air Pollution, 3 (in press).
Idem and Daaues, R. I. — (1960) Brit. J. Cancer, 14, 8.

Tobacco Manufacturers’ Standing Committee. — (1959) ‘Statistics of Smoking’
(2nd edition). London.

Wyers, H. — (1946) Brit. J. induslr. Med., 3, 177.

419

SMOKING AND CANCEE IN POLAND

J. STASZEWSKI

From the Institute of Oncology, ul. Gzenconej Armii 15, Glhvice, Poland

PART I vSTATISTIGAL DATA ON SMOKING AND “ TOBACCO TRACT ”

CANCER IN POLAND

Received for publication March 7, I960

The aim of this study is to discuss the results of the investigations on the
connection betu-een smoking and the appearance of some eancers. The investi-
gations were carried out on patients of the Institute of Oncology in Glivice
which is the largest centre treating tumours in Poland.

In the first part of the study a short presentation is given of the consumption
of tobacco in Poland and the appearance of “ tobacco tract ” cancer.

The view that smoking is a frequent cause of the appearance of lung cancer
and some other tumours is ever increasing; it is not however accepted by all.
That is why exhaustive investigations should be continued on the connection
between smoking and the appearance of cancer.

If a distinct connection between smoking and for instance lung cancer is
always found during the investigations in a variety of environments and in
different countries then this fact cannot be considered as accidental. iSuch

investigations should also make possible the recognition of the degree of the
association between smoking and cancer morbidity (chances of morbidity
depending upon the smoking habits) and explain what percentage of cases is
in the given environment connected with smoking. If this percentage is greater
in the environments \rith higher consumption of tobacco and lower in the environ-
ments in which act other carcinogenic factors — this will be the second serious
argument for the carcinogenic action of tobacco smoke.

The third argument would be the finding of a connection between smoking
and epidermoid cancer of the entire “tobacco tract”, of the whole mucous
membrane immediately in touch -svith tobacco smoke. It is difficult to imagine
that such a connection could be accidental. Interesting results should be
obtained by finding out the degree of this connection for cancer of various locali-
zations and by comparison of the data obtained with the localization of tobacco
tar m the respiratory tract of smokers (such studies have been started by Ermala
and Holsti (10.").^).

If. as some claim, the connection between smoking and “tobacco tract” cancer
IS not a causa .ye con..ection then it would have to be the effect of interdependence

tumoun,. Tl,c il.vcsligatio,, of

sl.ould enable one to detect such a factor. smoking and cancer

A. Consumption of tobacco in Poland

iu a;

(mostly cigarettes) after the second World ^YaT ^ ^consumption of tobacco

420

J. STASZEWSKI

Table I -Tobacco Consumption in Poland (Polish and Imported Tobacco]-
According to the Data Obtained from the Polish Tobacco Industry

Cignre ; in millions
Cigarettes (full smoking)
in millions (

Cigarettes (with mouth- (
pieces) : in millions J
Tobacco : tons ,

Snuff : tons
Choiring tobacco : tons
Total consumption per head
of the population (grammes
of tobacco products)

Note : In order to make the table smaller the data are presented for every fourth year. For 1938-14
hero are no data available; for lG4a~47 the data are not accurate and for that reason are not
presented here.

1925

1929

1933

1937

1949

1953

1957

52-2

78-0

36-5

28-9

24-9

22-5

31-2

■ 7,754-3

10,190-1-

f 3,762-4
b 3,842-6

3,747-8

3,951-0

19,881-2

809-6

30.088-4

428-3

41,539-0

1,012-2

11,242-2

13,011-3

10,927-0

12,583-2

973-0

342-5

389-7

445-6

314-0

252-8

92-6

95-4

603

8-0

4-8

7-5

692

549

562

849

1,216

1,491

Table II. — Tobacco Consumption in Poland in the Part Previously
under Russian Rule (Dolezal, 1922)

Year

Number of
inhabitants

Tobacco consumption
(pounds per head)

1829

4,137.634

0-72

1831

3,762,003

0-73

1832

3.914.6P5

0-94

1833

4,037,952

1-01

1834

4,059,517

0-83

1835

4.188,112

0-70

1845

4,798,658

0-79

1851

4,810,735

0-80

1855

4,647,454

0-93

1859

4,747,454

0-86

During the period between the first and the second Worid War mainly hand-
made cigarettes were smoked (in cigarette tubes or rolled in tissue paper). Since
the second World War hand-made cigarettes have been almost entirely given up
for factory-made cigarettes. The smoking of pipes and cigars, disappearing at
present, was rather popular during the period between the wars in the western
part of Poland (Pomerania, Poznan District, Silesia, i.e. in the previous Prussian
occupation^) but rarely met in the remaining parts of Poland.

Until recently there were no data on smoking habits of the individual smoker
or on the structure of tobacco consumption in Poland. It is estimated that
before the second World War the smokers constituted about 20 per cent of the
entire population and that after the war the percentage increased mainly by a
greater number of women smokers (personal communication from the Polish
Tobacco Industry).

In 1957 preliminary investigations were carried out concerning the smoking
habits and the structure of tobacco consumption in Poland (Staszewski an
Wisniewski, 1960). During these investigations the author collected data botli
on smoking habits (beginning, breaks, intensity and the manner of smoking)

‘ Up to 1918 the territory of Poland was devided for over 100 years by three occupants : Prussn',
Russia and Austria.

SMOKING AND CANCER IN POLAND

421

as well as on such background data as professional activities, the places of residence

{past and present) and the diseases the smokers had in the past.

Thus 2725 individuals of over 20 years of age were examined whose calling
at the Institute of Oncology in Gliwice was most probably not connected until
smoking as a causative agent: 1813 women (cancer and other diseases oi the
mammae-620, genital organs— 600, skin— 410, other organs which were not
exposed to the direct action of the tobacco smoke— 183) and 912 men (skin
cancer— 405, other skin diseases— 117, cancer and other diseases of the genitals
and mammary glands— 82, lymphatic system — 80, large intestine and rectum
71, skeletal system and soft tissues— 53, other organs— 104).

82-7 per cent of the examined men were smokers (present or past) who usually
started smoking at about 20; the percentage of the smokers increased with age
up to 60-70 years of life, the average intensity of smoking on the other hand
decreased with age. Among women there were 8*4 per cent smokers. They
had started smoking later and at various ages (average about 26 years old) ;
the percentage of the smokers increased up to 35 years of age and then distinctly
decreased. Differences were found in the consumption of tobacco between the
different professional groups in both sexes. No distinct differences in smoking
habits w'ere noted between men Ihdng in the cities and in rural areas, but among
women living in the rural areas there were markedly fewer smokers (1-8 per
cent) than among those living in cities (12-8 per cent). Pipe and cigar smoking
was met mainly among the population of Silesia (particularly among coal-miners
and foundry workers and markedly fewer pipe smokers among office workers):
the frequency of pipe smoking increased with age; it was not met before the
thirty -fifth year of life, nor among women. 91'5 per cent of smokers who smoked
cigarettes exclusively inhaled smoke as against only 34-7 per cent of those who
smoked only pipe and/or cigars and 68-3 per cent of mixed smokers.

Some other data obtained will be presented by the Tables of the second part
of this study.

B. “ Tobacco trad ” cancer in Poland

Up to 1950 the nomenclature of diseases and causes of death in Poland classified
tumours only into 2 groups; “ carcinoma and other malignant tumours ”, and
“benign tumours or not diagnosed as malignant”. Starting with 1951 there
is a more exact classification taking into account, among others, the group of
“ carcinoma and other malignant tumours of the organs of respiration ” Data
on the mortality caused by the tumours of this group are shown in Table III
c can see a distinct increase both in the number of deaths due to cancer of the
organs of respiration as well as an increase in their percentage in comnarison
with the total number of deaths or with the deaths due to all tumours There is
also a constant and distinct increase in the preponderance of men ^

Since lung cancer, constituting the largest number of natiVntc +i,;

» curaWo so for in only a small perccntele of tho oLS S fa FTP'

11.0 <Uag„osis a„,o„,„s L the aver^ to sft emi moSs it el t,
mortality in this case is only slightly smaller than morbiffity that

J>argc differences in the mortality (and thus in mnrLtVUfifx u t .

tants of the capital or industrial Silesia (Katom'ce Distritf mhabi-

d.stncts (as Kielce District lying more or leL half agncultural

aboot tat of aJfJJ thTSelrro The

422

J. STASZEWSKI

Table m.-~Eespirmonj Cancer MoriaUty {No. F-~99 N oynenclature of Diseases
ofStatlsH^ According to the Data Obtained from the Central Office

Percentage of deaths due to respiratorj- cancer

dumber of deatiis In comparison with

duo to cancer of total mortality

the respiratory organs , <

Year

Total

Men

—

Women

Poland

City of Katowice
Warsaw District

Kielce

District

1951 .

944

003

281 .

0-30

1-23

0-54

0-09

1952 .

1035

740

289 .

0-30

109

0-08

0-15

1.953 .

1278

924

354 .

0-48

1-58

0-73

0-28

1954 .

1472

1113

359 .

0-53

1-75

0-80

0-20

1955 .

1577

1190

387 .

0-00

1-70

0-89

0-25

19.50 .

1828

1418

410 .

0-73

2-23

1-24

0-32

19.57 .

2041

1530

511 .

0-70

2-37

1-10

0-39

In comparsioti with
mortality due to tumours

I —<

City of Katowice Kielc
Poland Warsaw District Distri

0-01

10-01

7-37

0-45

8-67

7-98

7-58

10-50

8-25

8-19

12-17

8-93

8-16

10-12

8-82

8-96

12-34

10-87

9-30

14-11

10-54

detection of tumours — difficult from the diagnostic point of view (as lung cancer).
The data presented for the whole territory of Poland should therefore be con-
sidered as lower than in reality and the mortality and morbidity rates for Warsaw
or Silesia are probablj'' the closest to the actual rates for the whole country.

Since 1954 there exists in Poland a regulation requiring the notification of
new cases of malignant tumours. It is not however strictly observed, e.g. in
1956 there were in Poland 20,409 deaths due to tumours (according to deatli
statistics) but only 17,796 newlj'^ detected cases of malignant tumours were
registered.

Lung cancer was, in 1956, the fourth in frequency of registered newly detected
cases of malignant tumours among males (617 cases), being after cancer of the
stomach (1444 cases), skin (822) and lip (747) and before cancer of the intestines
(296 cases), larjmx (270), oesophagus (220) and other organs.

The following are some data taken from the post-mortem statistics:

Novdcki (1931) claims that during the years 1896-1930 out of 30,957 post-
mortem examinations performed at the Institute of Pathological Anatomy of
the Jan Kazimierz University in Lwdwq the percentage of lung cancer (93 cases)
increased from 0-07 to 0-47. Out of a total of 2047 tumours the percentage of
lung cancer increased from 1-8 to 5-9. Syrek (1931) mentions that during 1901-30
in the Department of Pathological Anatomy of the Jagiellonian University in
Cracow, out of 28,455 post-mortem examinations 86 cases were found to be lung
cancer (67 men and 19 women). The proportion of appearance of this tumour
to the total number of post-mortem investigations increased during this penod
from 0-09 to 0-60 per cent, and in reference to the total number of cancer irom

1-36 to 7-18 per cent. , , . r q

Dabrowska, Poltorzycka and Trojanowska (1932) presented data rom
’Warsaw Departments in wdiich in the years 1911-31 out of 25,639 post-mor ems
performed in 212 cases lung cancer was found (169 men and 43 women). i
frequency of lung cancer in respect to the total number of i

from 0-5 per cent during the years 1911-13 to 1'4 per cent dunng 19 -
in respect to post-mortems due to malignant tumours from 5-7 to 1 • per •
Thus one can see that the increase in the frequency of appearance o o
cancer is apparent also on the ground of post-mortem statistics.

SJIOKIICG AND CANCER IN POLAND

423

Summary

The importance of epidemiological investigations into the problem of the
carcinogenic action of tobacco smoking is presented.

Certain data are given in reference to tobacco consumption and smoking
habits, concerning statistics on “ tobacco tract ” cancer mortality, and on post-
mortem statistics of lung cancer in P oland.

REFERENCES

Dabrowska, J., Pobtorzycka, S. and Tsojanowska, a.— (1932) GruzUca, 7, 259
Dolezal, F.— (1922) in ‘ Leopold Kronberg Warszawa.

Ermala, P. and Holsti, L. R.— (1955) Cancer, 8, 673-
No^vICKI, W. — (1931) PoUk. Gaz. lek., 10, 937.

Staszewski, J. and i\b§NiEWSKi, K. — (1960) Roczn. yauk roL, m press,

Sybek, a, — (1931) Polsk. Gaz. lek., 10, 995.

PART II.— TOBACCO SMOKING AND LUNG CANCER

Materials, Methods, Definitions

In more than 4 years between July 1954 and October 1958, 1031 men and
143 women were admitted to the Institute of Oncologj’’ in Gliwice m'th the
diagnosis or suspicion of lung cancer.

I interviewed these patients as to the smoking habits, professions and places
of residence in the same manner as the control group discussed in Part I of this
study.

In 298 patients (275 men and 23 women) diagnosis of lung cancer was con-
firmed by histopathological findings. Seventy-one patients (including 15 men
and 3 women rvith histopathologically proved lung cancer) were not intendewed
because of their too short stay at the Institute. There remain then 281 patients
(260 men and 21 women) intennewed with diagnosis confirmed histopathologicalty.
These patients are the subject of the present study. Our material comprises
over a half of the cases registered in Poland and possessing histopathological
confirmation, e.g. in 1956 there were 127 such cases including 68 cases from our
material.

The patients were dirdded into groups according to the histopathological
diagnosis: 1. squamous-cell carcinoma, 2. adenocarcinoma, 3. microcellular car-
cinoma, 4. undifferentiated carcinoma or a tj<pe histologicalty not determined
(foci carcinomatosi). The general characteristics of men belonging to these
groups as well as to the control group are presented in Table I.

Table II presents the smoking habits of men suffering from lung cancer, and
also the evaluation of statistical significance of the differences between Wem
ana the numbers characterizing smoking habits of the control group

^ year-and not less than an
vi.lu'J 1 tobacco a day— were considered as “smokers ”. The indi-

M.nictnt "'8^^.®ttes smoked pipe and/or cigars (each one smoked in a

smokem” ® ^ “smoker”) were called “mixed

424

J. STASZEWSKI

Table 1.—Ge7ieral Characteristics of 3Ien Suffering from Lung Cancer
and of the Control Group

Undiffer-

entiated

cnrcinorna

Nuinbor of individuals examined
Average age ....
Number and % of inhabitants
of towns

Number and % of inhabitants
of Upper Silesia

Number and % of office workers
Number and % of farmers

Squamous

cell

Adeno-

Micro-

cellular

histological
tj^ie not

Total

carcinoma

Control

carcinoma

settled

bronchus

group

137

260

012

50-5

52-S

50-7

55 -6

55-3

53-4

167

568

(62-0)

(60-0)

(75-0)

(04 -8)

(64-2)

(62-3)

104

192

641

(75-9)

(7o-0)

(75-0)

(69-0)

(73-8)

(70-3)

1)1

(2U2)

(15-0)

(21-9)

(22-5)

(21-2)

(12-2)

126

(5-])

(20-0)

(12-5)

(9-9)

(S'o)

(13.8)

Table II. — Smoking Habits of Men Suffering from Lung Cancer
and of the Control Group

Number of individuals examined
Number and % of smokers

Average intensity of smoking .
Average index of smoking
Number and % of bea%’}' smok-
ers (with the index over 300)
Number and % cf smokers

smoking only cigarettes
Number and % of smokers

smoking only pipe and/or
cigars

Number and % of smokers

inhaling smoke

Average duration of smoking
ha bits J

Undiffer-

entiated

carcinoma

Squamous

cell

Adeno-

Micro-

cellular

or Total

histological carcinoma
tvpe not of

Control

carcinoma cnreinomn§ corcinoma§

settled

bronchus

group

137

260

912

137

255

754

(lOO-O)t

(100-0)

(90-6)

(97-2)t

(98-1)*}:

(82-7)

17-2J

16-0

15-0

lo-9t

16-5*}

12‘2

666 -21

510-5

493-4

587 -6t

612-0}

406-3

133

235

447

(97-1)1

(85-0)

(82-8)

(S8-4)t

(92-2)}

{59-3}

117

218

5-^2

(S5-4)t

(75-0)

(79-3)

(91-3)t

(85-5)t

(73-2)

101

(l-45)t

(0-0)

(6-9)

(0-0)

(1-6)}

(13-4)

133

245

609

(9Ui)j:

(90-0)

(£6-6)

(95-6) t

(96-1)}

(SO-S)

38-7

32-9

31-0

38-1

37-1

32-2

* The calculations cf signifrcance have been taken from the study by Dr. Rozanowicz (unpublished),
t The difference with the control group is significant— exceeding over three times the standard
error of difference calculated in the equation ;

IJPjlIS

4 -

t The difference with the control group is e.xceedingiy significant, exceeding at least si.x times the
standard error of difference.

§ For adenocarcinoma, for microeellular cancer, and for average duration of smoking ha
significance of differences was not computed.

SMOKIXG AjSTD CAKCER IN POLAND

425

The duration of smoking ^vas determined after subtracting the breaks m

dailj is given as an average for the rvhole period of ^ """"I’'"''

that 1 cigarette = 1 g. of tobacco, and 1 cigar — 4 g. of tobacco. ^

The most frequently met classification of the individuals in reference o
smoWng habits is the one according to the intensity of smoking (^‘g;

1 package of cigarettes, or smoking more than 1 package a claj. etc.). In.
does not however take into account a verj' important factor of time, and ca ■
placing together into one group smokers who all smoke more tlian 1 package ot
cigarettes a day indeed-but one of them smokes for 5 years while another one
for 40 years. In order to avoid this and for the purpose of better cliaractcrizaf i on
of smoking by one number — giving some idea as to the intensity and the duration
of the smoking habit as well — ^the term smoking index uas introduced
(Staszewski, 1958). This index is the product of intensity of smoking multiplied
by the duration of smoking. If the indimdual smoked, for example, 12 cigarettes
a day on the average for 40 years then the smoking index would be 12 >: 40
= 480. This index multiplied by 0-365 gives the amount of tobacco in kilograms
smoked during the life of the individual.

Those who presented the smoking index over 300 were considered as “ heavy
smokers ”.

Individuals living steadily in a town or usually in a tomi and in the country
not longer than 10 years, are considered as “ town inhabitants

Individuals who lived steadily in Katowice or Opole Districts or not longer
than 10 years outside of these Districts are considered as “ inhabitants of Upper
Silesia ”.

For the classification of the individuals according to occupations, the occu-
pation performed the longest was taken into consideration.

Results

A. Men (Table 11)

The percentage of smokers was considerablj^ higher among the indiriduals
afflicted by lung cancer than in the control group. It is striking that there is
not one non-smoker among patients with squamous-cell carcinoma, who con-
stitute over oO per cent of patients with lung cancer.

xvaVr, '''■crage smoking index among smokers was 50 per cent higher in patients
with lung cancer than in the control group ^ paueius

<wragc duration of smoking depends on three \

the peixoirs o.xamincd (among patients uath Inncr^S factors. Average age of
higher than in the con rol CTmmV Irett ^ 2 years

snt,king. and the age of begi^ing

i-* re- easier

426

J. STASZEWSKI

Table III.— Smoking Habits of Women Sujfering from Lung Cancer
and of the Control Growp

Squamous

cell

carcinoma

Number of individuals examined 1

Number and % of smokers . J

( 100 - 0 )

Average index of smoking (cal- 540
oulnted for smokers)

Average index of smoking (cal- 540
culated for smokers and non-
smokers together)

Number and % of heavy smok- I
ers (with the index over 300) (100-0)
% of individuals examined 100-0
(smokers and non-smokers
together) being heavy smokers
Average intensity of smoking . 15

Undiffer-

entiated

carcinoma

Adeno-

Jlicro-

cellular

histological
tj-pe not

lotal

carcinoma

Control

carcinoma

settled

bronchus

group

1813

153

(9-1)

(20-0)

(75-0)

(28-6)

(8-4)

390

371 - 7

345-8

142-6

2-7

78-0

278-8

98-8

12-0

(0-0)

(100-0)

(60 -U

(66-7)

(13-2)

0-0

20-0

50-0

19-0

1-2

15-7

12-5

8-5

Among 137 patients ndtli squamons-ce]] carcinoma only 2 smoked less than
25 3 'ears; one — 14 j^ears (a j'ear after pneumonectomy performed in the thirty-
third }'ear of life carcinoma claroceliulare renis was found); the second — 21 years.
Among the patients -ndth adenocarcinoma 5 patients had smoked for less than
25 years, among the patients with microcellular carcinoma — 3, with undifferen-
tiated — 4.

The manner of smoking. — ^There were markedly fewer pipe smokers and/or
cigar smokers among the patients with lung cancer than in the control group.

Inhaling of smoke as well as smoking of cigarettes was much more frequent
among patients with lung cancer than in the control group.

The statistical significance of the differences in the consumption of tobacco
betAveen the patients having lung cancer and the control group are given in
Table II.

Histological subgroups . — Patients with squamous-cell carcinoma, who eoui-
prised over half of the patients discussed, present the liighest consumption of
tobacco. All without exception smoked tobacco and almost all of them were
“ hear^j' ” smokers (i.e. smoking index over 300), smoked cigarettes, and inhaled
smoke.

Similar, jmt a little lower, numbers characterize the second subgroup
“undifferentiated carcinoma or histological tjqpe not determined”.

Two remaining subgroups are small and it is difficult to drarv conclusions m
tills case. It seems that they smoke less than the patients wdth squamous-ce
carcinoma and among them pipe smokers and/or cigar smokers are met more
f^equentl 3 ^ On the other hand they smoke more than the examined indivi ua s
in the control group.

Other data.— 17-6 per cent of men with lung cancer had had pneumonia as
had 13-1 per cent in the control group (the difference statistical!}^ is o ™
ficance). Other diseases of lungs were rarely mentioned by the mm
in both groups.

SMOKING AND CANCER IN POLAND

427

Among the patients suffering from the lung cancer the division of h ood
groups was the following (for comparison the division of blood groups in Roland
fre Sven in brackets (Sablihski, 1959): A-41-7 per cent (37-1 per cent), B-
19-7^per cent (18-5 per cent), AB-6-3 per cent (7-6 per cent), 0-32-3 per cent
(36-7 per cent). Thus no connection was found between lung cancer and some

blood groups.

B. If omen (Table III)

The small number of cases makes detailed analysis impossible and onl}
allows a few general conclusions to be reached.

The numerical proportion of men to ■nmmen for the total number of patients
suffering from lung cancer amounted to 12-4 : 1, for adenocarcinoma onl}’'
1-8 : 1 and for the total of the remaining histological types 24-0 : 1.

The percentage of smokers, average index, and average intensity of smoking
were higher among women suffering from lung cancer than in the control group.

There is almost no difference in w'omen patients with adenocarcinoma in
reference to smoking and the control group, but in all the remaining histological
subgroups the consumption of tobacco is higher. The only w'oman with squamous-
cell carcinoma smoked and had the index of smoking above 300 (such index was
met in 1-2 per cent of women in the control group).

Discussion

A. Connection between smoking and lung cancer

Patients of both sexes suffering from lung cancer smoke markedly more
than the “ general population ” represented by the control group. Is it possible,
however, to compare these two groups! Do they represent the same population?

As can be seen from Table I, the differences in the social background of the
two groups are not great. Average age, the percentage of the inhabitants of
percentage of the inhabitants of Upper Silesia are very similar,
le merences in the professional structure are greater — but they could not
ser\e the explanation of the differences noted in tobacco consumption. In
T s'lf’groups of professions such a high index or percentage of smokers
r individuals suffering from lung cancer (average index of

nnrl tuf i ™ 364-4 among metal workers to 512-9 among railroad workers,
ainomr smokers from 70-3 among the office workers up to 89-1

urnfpRRinnc fhe control group in not one of the subgroups of

It mav be^D great as among the lung cancer patients),

ticallv hifrlilv that, as ascertained in our material, accurate statis-

is not ail connection betw-een tobacco smoking and lung cancer

«n.l q.acmi„£Ar connection-aratistioal

lhat our studies -irp ^ cannot give a decisive answer. It can only be stated

i'nportant role in the amiTshi smoking plays a most

Tho o f ^ genesis oi lung cancer.

, , ounce ion between smoking and lung cancer is quite distinct for patients

"f botli PCXes with Rnno ! canccr is quite distinct for patients

>'iibgrou]i as “uiidiffprpnf'pf carcinoma and with cancer included in the
if is, histologically not deter-

) distinct for microcellular carcinoma and adeno-

428

J. STASZEWSKI

carcinoma. In reference to the latter, conclusions cannot be drmvn unnn
asceitammg larger consumption of tobacco among men—due to the small mim^r

among women J.*

Our material is not sufficient to determine whether lung cancer possesses
any connection noth pipe and/or cigar smoking; if there were such a comection
It would be markedly smaller than in reference to cigarette smoking.

B. The chances of morbidity and the percentage of cases connected with smoHng

Relative chances of morbiditj^ in reference to lung cancer which the smoker
had m comparison with the non-smokers may be calculated for our material
by employing a formula given by Cornfield (1951);

d ~P2 )

Pi ' (1 ~Pi)

where A — relative amount by which the prevalence of lung cancer is augmented
by the attribute of smoking.

Pi = proportion of smokers among individuals examined with lung cancer.

Pi — proportion of smokers among individuals examined in the control
group.

Using this formula we find that a smoker possessed about 10-fold greater
chances of becoming afflicted by lung cancer than a non-smoker.

Tiie proportion of cases connected with smoking null then be :

^ j

y = - X Pi ~ 88-2 per cent.

And thus in almost 90 per cent of male individuals we found a connection
between smoking and lung cancer.

The same problem may be discussed in another way. Let us calculate how
many men ivith lung cancer we would expect to meet if smoking among the
entire male population were the same as among women and if there were a close
correlation between the appearance of lung cancer (except adenocarcinoma) and
high tobacco consumption represented by a higher index than 300. Such an
index appeared in 49-1 per cent of the total number of examined men and in
1-2 per cent of the total number of women in the control group — proportwn
41 : 1. And therefore a decrease in the frequency of appearance of a higher
than 300 index by 41 times avouM also bring about a decrease in the number of
men afflicted by lung cancer. We Avould then have not 240 but only 6 men suffer-
ing from lung cancer of a t3^e other than adenocarcinoma and therefore a
number as in women (10 cases after subtracting cases of adenocarcinoma).
elimination of smoking as a carcinogenic factor would also decrease a little the
number of 26 cases of lung cancer in men and 21 cases in women. It woim
result then that smoking played (Avith the above foundation) the mam ro e nx
over 90 per cent of cases of lung cancer among men (>234/260) and m
per cent of the total number of cases (>234/281). The numbers obtained b)

both methods are similar.

SjrOKIICG AJfD CA2fCER IN POLAND

429

Conclusions

Our obserA^atious prove that in Poland there is also a distinct comiection
between cigarette smoking and the appearance of lung cancer The connection
is obsen*ed in men as well as in -women— most distinct^ for squamous-cell
carcinoma. Contrary to the findings of other authors the connection oi smoking
■noth microcellular carcinoma has been less distinct in Poland than mth adeno-
carcinoma; both these subgroups were, however, too small to draw far-reaching
conclusions.

The markedly more frequent appearance of lung cancer among men can be
easily explained when accepting that smoking is frequently the cause of cases
presenting this neoplasm.

The period from the moment of starting smoking to the appearance of
symptoms of lung cancer was usually longer than 25 years (on the average it
amounted to 37-1 years). Therefore a conclusion can be reached that in studying
dependence between smoking habits and morbidity due to lung cancer in the
examined population one should take into consideration consumption of tobacco
during the last 30-40 years and not the present consumption.

It is evaluated that men smokers possessed about 10-foId greater chances
of becoming afflicted by lung cancer than the non-smokers and that smoking
has been connected ndth about 80 per cent of the total number of lung cancer
cases.

Summary

Results of a retrospective study conducted in Poland on tobacco smoking and
lung cancer are presented. This study shows a very distinct correlation between
cigarette smoking and lung cancer.

It is considered that men smokers possessed about 10-fold greater chances of
Incoming afflicted with lung cancer than non-smokers and that smoking was
connected -^nth about 80 per cent of the total number of lung cancer cases.

Tile long period from the moment of starting smoking to the appearance of
symptoms of cancer points to the necessity of taking into consideration tobacco
consumption during tlie last 30-40 years during the study of correlation between
smoking habits and morbidity due to lung cancer.

The mdex of smoking (intensity x duration) seems to be a better criterion
ot classiiication of smokers than intensity of smoking.

REFERENCES

( ortxi iKLD. . 1 . — J. Cancer Inst. H 1269
SMuaxsKi. Pol. med. }('%., 13, 63

STAszr.w.sKi. .1.— {1!W8) XoiL-ohcory, 8, 51.

PART III.~CAxNCER OF THE “TOBACCO TRACT”

(EXCLUDING LUNG CANCER) IN IIEN

® ‘Ob-- --

of tho lip. 83

430

J. STASZEWSKI

coiK^tions of the oral cavitj', 19 -jvith carcinoma of the tonsils, and 207 ivith
carcinoma of the larynx.

A group of 912 men over 20 years old examined in the first half of 1957 whose
reason for calhng at the Institute of Oncology was most probably not connected
mth smolang serves as a control group (group “ G ”). This group is described
m general outlines m the first part of tins study which discusses tobacco con-
sumption in Poland.

Additional comparison group (group “ D ”) for patients with carcinoma of
Jip mil be represented by 200 farmers afflicted by carcinoma of skin.

The manner of collecting data both for the group of examined individuals as
u ell as in both control groups and the defim’tions “ smokers ”, ” heavy smokers ”,
“ index of smolang ”, “ the inhabitants of towns ”, etc. are as described in Part If
of this study on lung cancer.

Results and Conclusions

In Table I some general data are presented in reference to patients afflicted
by cancer of various sites, and in Tables II and III data on their smoking habits.
IVe shall discuss cancer of each site in turn.

Table I. — General Characteristics of Men with Cancer of the “ Tobacco Tract ”

and of the Control Group

Carcinoma

Oral cavity

and

/" ' ■

precancerous

Pre-

Carcinoma Carcinoma

conditions

cancerous

of the

Control

of lip

Carcinoma conditions

Total

tonsils

larjTix

group

ICumberofindividunts

391

207

912

examined

Average age

S5-S

57-9

53-0

6G-4

62-5

56-4

53-4

Number and % of in-

114

138

568

habitants of tOViTlS

(28-9)

(65 -5)

(60-0)

(63-9)

(78-9)

(6B-7)

(62-3)

Number and % of in-

217

149

041

habitants of Upper

(35 -1)

(72-4)

(G8-0)

(71-1)

(C8-4)

(72-0)

(70-3)

Silesia

Number and % of

111

office -srorkers

(4-1)

(19-0)

(20-0)

(19-3)

(31-6)

(12-6)

(12-2)

Number and % of

12G

fanners

(32-0)

(13-8)

(8-0)

(12-0)

(5-3)

(IM)

(13-8)

1. Cancer of lip

In 387 patients changes concerned the lower lip and in 7 the upper lip. Histo-
pathological diagnosis of squamous-cell carcinoma was positive in 306 patients,
uncertain in 41 patients (probably carcinoma, suspicions of carcinoma), and
in 47 patients only precancerous conditions were found (hjqierker^osis et pro-
liferatio epithelii, paratjqiia epithehi, papilloma, leukoplakia). The patients
in question were divided into 2 subgroups; farmers and non-farmers-smce
cancer of lip is particularly frequent in farmers, which suggests the pertinence oi

“IhriatToSSS the tobacco cooaocption to each of ‘j;--
presented in Table II. Before discussing it, however, a few words
to the influence of atmospheric factors on the appearance of cancer ot tip.

SMOKING AND CANCER IN I’OLaND

432

J. STASZEWSKI

. of non-farmers the percentage of individuals exposed because

of their occupation to the action of atmospheric factors was markedly higher
than in the control group “ G There were in this group for instance more
workers employed in the building industry (16-4 per cent in comparison with
9-0 per cent in the control group after subtracting the number of farmers) and
less office workers (5-9 per cent in comparison wdth 14-1 per cent in the control
group) or inhabitants of tomis. This type of connection with the exposure to
atmospheric factors ivas not found in the other cancers discussed later on. It is
worth mentioning that among the patients with cancer and precancerous con-
ditions of lip, farmers constitute almost one-third of the total number of patients
(126 out of 394), wliile in the control group only about one-seventh (126 out of
912). The farmers are really particularly exposed to the aetion of atmospheric
factors. It can be supposed then that these factors (probably the ultraviolet
rays of the sun spectrum) play a large role in the appearance of precancerous
conditions and cancer of lip.

Let us now take into account the tobacco consumption among the patients
mentioned above.

The percentage of smokers, average intensit}^ of smoking, average index of
smoking and the percentage of heavy smokers (i.e. 110111 the index of smoking
above 300) were markedlj’^ higher among patients with cancer and precancerous
conditions of lip than in the control group “ C ”. This is observed in all the
subgroups of Table II (only in two small subgroups the percentage of lieaiy
smokers was a bit lower than in the control group). The differences compared
iidtli the control group were larger for non-farmers (statistically significant) than
for farmers for whom these differences w'ere statistical] 3 ’ of no importance or
on the boundary of significance.

Let us compare now' the farmers suffering from cancer and precancerous
conditions of lip not with the control group “ C ” but ivitli the additional group
for comparison (“ D ”): 200 farmers wdtli cancer of skin, not a large group really,
but more corresponding in reference to the mode of life, conditions of w'ork, etc.,
and thus representing better the consumption of tobacco among the fanners.
Such a comparison brings into prominence higher tobacco consumption amoug
farmers wdth cancer and precancerous conditions of lip — e.g. the percentage of
smokers among them is distinctlj' liiglier than in the additional group (“ D ”)
for comparison (the difference here exceeds 3-7 times the error of difference).

The manner of smoking. — Differences in reference to percentage of smokers
using only cigarettes, percentage of smokers using only pipe and/or cigars, and
the percentage of smokers inhaling smoke wmre statistically not significant,
however among patients mth cancer and precancerous conditions of lip there
were a few more cigarette smokers and smokers inhaling smoke than in the contro
group. This is contrary to the results obtained by other investigators who hare
stated that the connection between cancer of lip and smoking is particular y
distinct in smokers using a pipe and/or cigars. The reason for this drsagreemen
is not clear. It may be caused for instance by a small number of pipe and cigar
smokers among our patients — studies carried on larger material could perhaps

explain that.

Chances of morbidity and the percentage of cases connected with smoking wull be
calculated similarly as for lung cancer in Part II of this study.

SMOKIKCt AKD CAXGEU IX rOLAXB

Relative chances of cancer and precancerons conditions f
hasl comparison ndth a non-smoker amount to. after Cornfield s (Ifi.,1) fonnnla.

A -

0-034

n-S27

(I - 0-S2T)
n — 0-034^

2 - 00 .

The percentage of cases connected with smoking amounted to.

y = 61-S per cent.

Analogic computations for the subgroups of farmers when compared with the
additional comparison group “D” give:

_ 0-905 (I — 0-755) _
~ 0-755 • (1 - 0-905)

7 / = 61-2 per cent.

Concluding, among our patients we find a distinct connection between the
appearance of carcinoma and precancerons conditions of lip, and tobacco smoking
and exposure to the action of atmospheric factors. Pipe and/or cigar smoking was
not found to be more connected witli the discussed diseases than smoking of
cigarettes. The smoker had 3 times greater chance of becoming afflicted than
the non-smoker. About 60 per cent of cases were connected with smoking.

2. Cancer of the oral cavity

In 58 patients the diagnosis of squaraous-cell carcinoma was proved by lii.sto-
pathological examination and in 26 patients there were found only precancorou.s
changes (leukoplakia, suspicious epithelial proliferation, papilloma). Difforoncos
in the smoking habits of both of these small subgroups were not great (Table III).

The percentage of smokers, average intensity of smoking, average index of
smoking and the percentage of heaw)’^ smokers were significantly higher among
patients suffering from cancer and precancerons conditions of the ora! cavity
than in the control group. But the differences in the manner of smoking were
not significant from the statistical point of view. The percentage of those
smoking only pipe and/or cigars and not inhaling smoke was hoAvever a little
lugher among the discussed patients. Tliis is in agreement with the statement
by other investigators that there is a more distinct connection between cancer
of the oral cavity and smoking pipe and/or cigars than with cigarette smoking.

The relative chances of a smoker to become afflicted b 3 ’ cancer and precancerons
conditions of the oral cavitj' amounted to;

j __ 0-988 (1 — 0-827)

0-827 ■ (1 — 0-9SS)

17-2.

Tlus number may be considered as only a near estimate because with the small
number of non-smokers it is very dependent on chance (a slight change in the

denoSor? markedly changes the numbe® in the

denommator). The percentage of cases connected with smoldng amounted to:

y = 93-1 per cent.

Concluding among the discussed patients there was a dhfinot ^ +-
beuveen and cancer and prfcancerona conSiom

434

J. STASZEWSKI

Table III. Smoking HabiU of Men with Cancer of the Oral Cavity, Tonsils and

Larynx and of the Control Group

Cnr-

cinoma*

Number of individuals examined 58
Number and % of smokers . 57

(98-3)

Average intensity of smoking . 15- 1

Average index of smoking (calcu- 544-7
lated for smokers)

Number and % of heav}’ smokers 47
(with the index over 300) (82-5)

Number and % of smokers smok- 39
ing only cigareties (S8'4)

Number and % of smokers smok- 12
ing on]3' pipe and/or cigars (21-1)

Number and % of smokers inbal- 40
ing smoke (70-2)

Oral cavity

Pre-

cancerous

condi-

tions*

Total

Carcinoma Carcinoma
of the of the

tonsils* larj-nx

Control

group

207

912

200

754

(100-0)

(98-8)§

(100-0)

(99-o)§

(82-7)

12-2

14-9

15- Ot

16-4

15-7?

492-4

535 -4|

600-8

585 -3§

406-3

447

(80-0)

(81-7)J

(68-4)

(88-8)

(59-3)

182t

552

(88-0)

(74-4)

(68-4)

(88-3)

(73-2)

101

(8-0)

(17-1)

(15-8)

(1-9)

(13-4)

I99|

609

(88-0)

(76- C)

(78-9)

(96-6)

(80-8)

Note : The calculations of significance have been taken from the study bj-^ Dr. Rozanowicz (per-
sonal conmmunication).

* For this column significance of difference was not computed.

t The difference vith the control group is significant — exceeding tvo to tluee times the standard
error of difference calculated in the equation

S.E. = £ii;

I The difference with the control group is distinctlj- significant — exceeding three to six times flie
standard error of difference.

§ The difference with the control group is exceedingly significant — e.vceeding at least six times
the standard error of difference.

The smoker had about 17 times greater chance of becoming afflicted tlian the
• non-smoker. About 90 per cent of cases were connected with smoking.

3. Carcinoma of tonsils

In tliis small group in all cases the diagnosis of squamous-cell carcinoma was
confirmed bjr histopathological findings.

All the patients were smokers. Average intensity and average index ol
smoking and the percentage of heat^^ smokers wmre markedly Ifigher than in
the control group, but the manner of smoking did not tfiffer in both groups.
Due to the small number of patients with carcinoma of tonsils statistical appraisa
of these correlations is not possible.

4, Carcinoma of larynx

In all the patients the diagnosis of squamous-cell carcinoma was confirine
by histopathological findings. Extrinsic larynx cancer was also mcluded here
There were only slight differences in the smoking habits of the patients
cancer of the larynx of various localizations.

SMOKING AND CANCER IN POLAND

The percentage of smokers, average intensity of smoking, average index of
smoking^and percentage of hea\- 3 ' smokers were markedly Ingher among these
patientf than^n the control group (the dilferenccs being statistically eMremcl^

^'^he pLcentage of smokers using only cigarettes as well as the percentage
of those inhaling smoke was among the patients with cancer of larynx markedly
higher than in the control group; these percentages were also higher than in
patients with cancer of lip, cancer of the oral cavity, or tonsils— and very .similar
to the percentages obsen^ed in lung cancer (see Part II of this study).

The relative chances of morbidity for smokers amounted to :

0;9^5 (1 - 0-S27) ^

~ 0-827 ■ (1 — 0-905)

The number obtained above is only of orientation significance bccau.se of the
very small number of non-smokers — it would greatly change if for instance
instead of one there were two non-smokers. The obtained number however
proves that the smoker had many more chances of becoming afflicted by cancer
of larynx than the non-smoker.

The percentage of cases connected with smoking amounted to:

y = 97-1 per cent.

Concluding, among the discussed patients cancer of lar^mx is verj’ distinctly
connected with smoking and particularly witli cigarette smoldng and inhalation
of smoke. The smoker had about 40 times greater chance of becoming afflicted
by cancer of larynx than the non-smoker. Over 90 per cent of cases afflicted
by the said tumour were connected with smoking.

5. Women

Among 31 examined women, not included in the Tables (13 uith carcinoma
and precancerous conditions of lip, 3 with carcinoma of the oral cavity, 2 of the
tonsils, and 13 with carcinoma of larjmx) 7 were smokers (2 with carcinoma of
lip, 1 carcinoma of the oral cavity, and 4 carcinoma of larjmx) in whom the
average index of smoking amounted to 307-9. These numbers are not sufficient
to draw far reaching conclusions. They show however higher toliacco con-
sumption among women with the discussed tumours than in the control group of
1813 women (discussed in Part I of this study, where 8-4 per cent were smokers
and the average mdex of smoking among the smokers amounted to 142-6).

jjiscussion

population! ^ ^ comparable? Do they represent the same

witil'ls of h or‘ o b't'veen pationb

.nee f,., oLa “

436

J. STASZEWSKI

of Upper Silesia as well as occupational structure of these groups are similar
The reason for the difference in structure of the group of patients afflicted bv
cancer of tonsils may be the small number of patients in this group. Among the
patients suffering from cancer of lip, the previously discussed differences in the
occupational structure and first of all a large percentage of farmers is noted. The
farmers Avere placed in a separate group haAong an additional group “ D ” for
comparison.

In none of the occupational subgroups of the control group higher percentage
of smokers than 80-1, or aA^erage index of smoking higher than 512-9 Arere noted,
and the differences in the smoking habits betAveen the inhabitants of toAAms and
rural areas Avere not great. The differences in the tobacco consumption betAA-een
the control group and the patients discussed cannot therefore be explained bj-
sampling error. The CAmluation of statistical significance of these differences
speaks against their accidental appearance. It should be accepted then that
there exists a distinct coimection betAA^een the appearance of the discussed neo-
plasms in Poland and tobacco smoking.

Summary

Results of a retrospectiAm study conducted in Poland on tobacco smoking and
cancer of lip, oral cavity, tonsils and larynx are presented. This study sliOAAxd
a distinct correlation betAA'een tobacco smoking and the above-mentioned cancers
among men.

The relative chances of morbidity of smokers and the percentage of cases
connected AAuth smoking are computed.

The number of AA-omen examined Avas small but even among them tobacco
consumption aa'us found to be higiier than on the average.

The author aAmils himself of the opportunity to expre^ his gratitude to
the Director of the Institute of Oncology in Glivdce— Dr. SAviecki, M.D.— for
encouragement and valuable advice, and to Jlr. D^bogorski, Master of Lavs,
Director of the Polish Tobacco Industry, and to his co-AAnrkers: Mr. Trojan,
Doctor of Chemistry, li'Ir. Trzcinski, Doctor of Agricultural Sciences, Mr. Compa a,
Master of Chemistry, Mr. Wierzba, Master of Agricultural Sciences— for thor
friendliness AAdiich rendered possible tliis study.

REFERENCE

CoENFiELD, J. — (1951) J. nai. Cancer Inst., 11, 1269.

ON THE BIMODAL AGE DISTRIBUTION OF
MAJHIAEY CARCINOMA

F. DE

WAAED,* J. W. J. DE LAIVE akd
A. BAAEDERS-VAE HALEWLIN

From the. Department of Medicine, Umvereitu Hoepilal and the
Diahonessenhvis. Utrecht. Holland

Beceived for publicntion Mny 10, 1900

Ix 1930 — shortly after his death — a book wTitten bj" von Pirqiiet was pub-
lished This treatise gave an analj'sis of the age distribution of a large number
of malignant tumours, the mortality statistics having been obtained from tlic

British Registrar-General. . -i .■

One of the interesting facts presented was a bimodal type of age distribution
of mammary carcinoma ; it was shorrm that the distrilnition was probably built
up by two separate distributions, one harnng its highest frequency at about 50
years of age, the second tjqje (a “ Spatkrebs ”) being most frequent at about 70
years.

After World War II attention to this peculiar feature was drawn again by
Jacobsen (1946) and by Clemmesen (1948) who had been working with morbidity
figures of the Danish Cancer Registration. From many sides confirmation of
the bimodal tjyie of age distribution was published : Andei-son el ah (ItloO) gave
morbidity figures from Connecticut, U.S.A., Maisin and Langerock (1955) pre-
sented graphs of Danish, Swedish and French material at the Congress of Geo-
graphical Fathologj’^ held at Washington ; at the Perugia Symposium on mammary
cancer Denoix (1958) showed the French data in full, while Desaivc, Lavigne
and Adrianne (1958) gave figures from Belgium. Further confirmation came from
Ficke and Reiszig (1958) from the German Democratic Republic and from
Pedersen and Magnus (1959), the latter paper being an official publication of tlie
Norwegian Cancer RegistrJ^

Ii\ the material of the Dutch Cancer Registry we found a bimodal type of non
distribution too. Fig. la shows an age specific frequency cunm of morbidity,
and Fig. lb presents an age specific curve of mortality, computed from data of
the Central Bureau of Statistics. These curves run upwards steeply' but show a
diminution of this trend between the 45th and 55th year, namely in tiie period
of onset of the menopause. This phenomenon could be interpreted by assuming
that at menopausal age the curve of the first kind of mammarj^ cancer (tliat of
reproductive^ age) decreases sharply, while the curve of the second kind (the
deeSasr^' increased so much that it already compensates for that

However the typical bimodal age distribution is not found in every statistical
material. The figures of Phillips and Owchar (1957) from eight countries are
not c„„s,dered here, because they have been related to a staMar^^raaMo”

438

de WAABD, DE LAIVE ANB BAA^TDERS-VAN HALEWIJRt

from abroad (Canada). But the curves which can be constructed v-ith Hip
figures of Stocks (1959) show that in the metropolitan areas of the USA no
significant retardation of the steeply increasing curve of age specific morbldiv

“ contrast to the Scandinavia^
^ ^ possible explanation of this fact later.

Several authors have been wondermg what this bimodal age distribution
actually means. Gan we speak of two types of mammary cancer ? And in
winch respect do these types differ ? Is their cause also different ?

Fig- la.— Age-specific inorbidit 3 ^ (per 100,000 women) of maramarj^ carcinoiDa in the JsTether-
lands. 3031 cases diagnosed during 1957 and 1958 and collected bj”" the Central Cancer
Kogisfcrj’', Amsterdam. It is estimated that about 70 per cent of all cancers are being
registered at present; no correction for this has been attempted.^ Population figures
supplied hy the Central Bureau of Statistics, The Hague (population, at December 31,
1950).

Maisin and Langerock (1955) made some suggestions as to the nature of the
two types. They mentioned infer alia that from animal experiments evwence
had been obtained that in the genesis of mammary cancer of older age an adreiia;
dysfunction played a part. Their compatriots Desaive, Lavigne and Adnanne
(1958) went a step further at the Perugia Symposium, postulating irithont any
reserve that the type of mammary carcinoma of reproductive life was due to a
dysfunction of the ovaries, and the postmenopausal typie to a d3’^sfunction o i

Ficke and Peiszig (1958) were more cautious, but they too drew attention o
the decreasing incidence of the younger type of mammary cancer wnen

AGE BISTRIHUTIOX OE MAMMAEY

CAKClKOMA

XS; rottX «vc ccS tSut.:

Sl»nd (M<n„bocU »,„. Boot, .P.,»).

Fig. lb. — Age-specific mortality (per 100,000 -n-omen) of mammary carcinoma in the Xether-
lands. 3191 death certificates registered during 1957 and 1958 by the Central Bureau of
Statistics, The Hague. N.B. : It is for the greater part about other women than in the
graph of morbidity. The anomaly in the curve between 45 and 55 years is less than in
the curve of Fig. la probablj- due to the longer and inconstant period between onset and
registration of the malignancies.

In laboratory animals early bilateral adrenalectomy is equally effective as early
castration in lowering the incidence of spontaneous mammarji carcinoma (Shimkin
and Wyman, 1945). There are many indications that the therapeutic significance
of bilateral ovariectomy and adrenalectomy is based on the elimination of oestro-
pns. If it should be possible to distinguish by clinical or laboratory means
between ovarian and adrenal oestrogens the hypothesis could be tested that the

s: .Ss sreSr'"

Ontological work of Bruinsma and de Waard 119591 ^ppme
d, sting., ish bettveen these tn-„ t,T,e.. These antSlSJ tre^demio”

440

de waard, de lah^e and baanders-van HALEB^IJN

logical considerations concerning endometrial cancer wondered f

f A f difference between these groups\“s

g 0 the diabetic patients Tinth obesity and/or hj^pertension. IiUherase of

Fio. 2. — ^Age specific morbidity of mammary carcinoma per year per 100,000 women, after

Stocks (1959, Table 4).

patients with obesity and/or lij'pertension without diabetes oestrogenic smears
were frequent!}’’ encountered too. Because of the limited number of diabetic
patients -without obesity or hj'pertension it was impossible to draw any conclusions
about a possible difference between these patients and the control group. Of
significance were the findings of oestrogenic smears in 17 castrated diabetics. The
authors concluded that the oestrogens responsible for these effects were probabh
of adrenal origin. • f

De Waard and Baanders (to be published) made a cjdological inrestigarion o
the urinary sediments of more than 100 women over 55 years of age from t le
population of Utrecht, Holland, who had been invited to serve as a normal control
group in a genetic investigation. There exists a c}i;oIogical parallelism between

age distribution of mammary carcinoma

■Ml

the vaginal smear and the urinary sediment (Lcncioni, 1 nr)3), permitting endocrine
Valuation of the latter after staining wth Shorr's tnchromc stam. J he results
of this investigation confirmed and completed those of Bnunsma and do an

a959l by shov-ing clearly the influence of hypertension

frequLcy of oestrogenic pictures in women with ovaries as well as m those
vdthout. Moreover, a study was made of glucose tolerance m the non-o lesc.
non hjTiertensive subjects. It was shown that the few women of his subgroup
presenting a certain oestrogenic activity had a decreased glucose tolerance. 1 he
Lthors concluded that the comhined groups of postmenopausal woincii ^ith
obesity hjmertension and a decreased glucose tolerance contained all tliosc
exhibiting oestrogenic activity cj-tologically. This condition of continuous
hormonal activity was called : adrenal oestrus.

PLAN OF STUDY. MATERIAL AND METHOD

Based on this cjdological evidence for adrenal oestrus and on the epidemio-
logical fact of a bimodal age distribution of mammary cancer the following plan
of stud5' was made ;

Of a number of patients with mammarj^ carcinoma we investigated :

Weight and height.

Blood pressure.

Glucose tolerance.

If obesity, hj'pertension or a decreased glucose tolerance were present it ivas
assumed that adrenal oestrogen would have been present which could have
promoted the carcinogenesis in the mammarj’- gland. If none of these pathological
signs was found it was supposed that any carcinogenic influence of oestrogens
would have come from the ovaries. Of the “ adrenal ” as well as of the “ ovarian ”
type of patients with mammarj'- cancer an age distribution could be made ; if
these age distributions coincided with the distributions found by epidemiologists
in their morbidity statistics an argument would be provided for the correctness
of the hypothesis.

The material obtained from 108 patients was collected in six different hospitals.
The patients belonged for the greater part to the population of two large tomis.
Only cases of recent onset (less than six months between first diagnosis and oiir
investigation) in which the malignancy had been confirmed by the pathologist
were studied. Cases with distant metastases and those who were or had been
treated endocrinologically (with hormone preparations or by surgical intervention)

The criteria for classification according to relative weight, blood pressure and
glucose tolerance were the foUowing : a body weight more than per eJn^
above Ideal eight was caUed obesity ; we used the Table of Ideal Weights

Wedervang (1957). In lajdng down the criterif for

fSrplacrtL^irf ^-^tohe pres^re rtt

. p..rc,y arbitral- „„e. Ho.evar. we fo;„i’d3^riuSblf

442

DE WAARD, DE LAIVE AND BAANDERS-VAN HALEWIJN

h 3 ’pertensive cases those with a diastolic pressure of 100 mm. Hg or more if their
Bj-stolic pressure was at least 150 mm. Hg, and those with a systolic pressure of
1 /O mm. Hg or more irrespective of diastolic pressure.

The glucose tolerance tests were carried out either pre-operativeh^ or more
than 14 days post-operatively in order to avoid the effect of surgical stress The
amount of glucose given urns 50 g. by mouth ; the blood sugar estimations were
performed in 73 cases according to the method of Hagedorn and Jensen and in
35 according to Polin and Wu.* In judging the blood sugar ounces attention
was given to both components composing a diabetic tj-pe of curve separately :
1 . blood glucose level increasing too much, 2. level not decreasing to original (fasting)
value within 2 or 2J hours. Ad 1. we considered the level to be pathological,
if at least two values above ISO mg. per cent or one value above 190 mg. per cent
U'as reached. Ad 2. we fixed a dividing line between normal and abnormal after
making frequency distributions of the difference betu'een the 2- or 2|-hour values
and the fasting values. It was seen that also at higher ages a decrease to below
the fasting value often took place, and that a suitable boundary between normal
and abnormal was to be found in the region of 2- or 2|-hour levels Ijdng 10-20 mg.
per cent above the fasting values. We chose finally as the dividing line : 20 mg.
per cent above the fasting value, considering curves with differences of 15-20 mg.
per cent dubious ones.

RESULTS

Based on the above criteria we divided the patients into two groups : one
without anj' pathological feature of the triad — obesity, hj^pertension or a decreased
glucose tolerance, the second with at least one of those features. Tliree women
who W'ere not obese nor hj'pertensive, but who exhibited a dubious blood sugar
curve were classified separately in Table I and omitted from Fig. 3 which thus
presents the result of our separation of 105 cases. In this figure which is a
graphical condensation of Table I two frequency distributions are seen nith
modes in the 45-50 and the 60-64 age classes respectively, wliich show a striking
similarity to tlie distributions expected by epidemiologists. This does not prove
that our hjqrothesis is correct, but it gives definite support to it.

An important question wduch has to be investigated is : does one in separating
any female population according to the presence or the absence of obesity,
hypertension and decreased glucose tolerance perhaps find two age distributions
similar to the distribution of mammary cancer patients ? It is well knorni that
the features of tlfis triad are much more frequent later in life than in the repro-
ductive period. r j. f I.

We have investigated tliis point w’ith care, making use of data of a statistica -
genetic study designed by two of us (not yet published). The control ^^up
needed for this study was obtained by taking 1000 cards of women ^
files of the Population Registry of the town of Utrecht (250,000 inhabitant^.
This wms done at random apart from a certain age distribution, ihese women
■u'ere invited to an interview about cancer in their families, together wn i a J
physical examination including measurement of weight, height, bloo
and examination of the urine. Almost 60 per cent of these women co-operateu ,

* As judged by frequency distributions of the fasting levels the values obtained by the forme
method are about 5 mg. per cent lower than those obtained by the latter.

443

age DISTRIBUl’ION OF MAMMARY CARCINOMA

30 35 40 45 50 55 60 65 70 75 80 85

AGE

Fig. 3. — Separation of 105 cases of mammary carcinoma into two groups (sec text).

444

DE WAAED, DE LAIVE AND BAANDEES-VAN HALEWIJN

an anatysis of tlieir addresses revealed that the living quarters of the higher
educational classes were represented slightly better than the other ones, llakinff
a comparison with a group of mammary cancer patients tins does not seem to
be a draw-back, because it is knoAvn that patients with tliis tjqre of cancer consti-
tute also a slight selection among the liigher educational population groups.

The investigation provided us with the data of 571 women. They \rere
divided into two groups according to the same criteria of obesity and hypertension
as the 108 patients with mammary cancer (it Avas impossible to perform a glucose
tolerance test in all of them). In view of the shape of the lustograms of Kg. 3
Ave AA’ere interested in tire question whether obesity and hypertension (occurring
singly or combined) Avere more frequent in the postmenopausal cancer patients
than in the postmenopausal controls. Table II shows that this was found indeed.
IVithin both groups there exist small differences betAveen the age classes Avdiich
are, hoAvever, far from significant (y"-test). Thus it is permissible to combine the
different age classes aboA^e 55 years and to summarize as follows : in the mammary
cancer group 42 of 59 AAmmen are obese and/or hj'pertenshm, that is 71 per cent.
In the control group 174 of 322 women haAm obesity and/or lijyertension, that is
54 per cent. Although this latter percentage is liigh too, the difference from the
mammary cancer group is significant (hjqjergeometrical distribution, P < 0-02).
If the criterion for hypertension is modified in such a way that only blood pressures
of at least 170 mm. Hg systolic fall into the pathological group, the difference
betAA’een the cancer group and the control group becomes even greater (P < 0-01).

Table II. — Relative Frequency of Obesity and Hypertension {Occurring Singly or
Combined) in Mammary Cancer Patients and Controls Over 55 Years of Age

Age

llammary cancer
group

Control group

55-59

60-64

65-59

70-74

75-79

80-84

Total

Number of women

. 15

4 .

Number of women
with obesity
and/or hj-per-
tension

. 9

Number of women

. 78

19 .

322

174

Number of women
with obesity
and/or hj-per-
tension

. 40

12 .

Thus the conclusion seems to be justified that mammary carcinoma after the
menopause has a preference for the obese and the Iwertensive occurring aimcsr
exclusively in women Avith obesity and/or hypertension f
glucose tolerance, conditions wliich are associated with oestrogenic aetn }

adrenal origin.

DISCUSSION

The direct CAudence of a connection between adrenal “ggnt'

and a decreased glucose tolerance in older people not yet being p } P
some indirect evidence may be wanted.

AGE DISTRIBUTIOK OP MAMMARY CAUCIXOMA

(-15

It is kno^n^ that older patients witli diabetes niellitns with or without obesit.t
who do well on a dietarj^ regime alone or in conjunetion witli oral adininist ration
of^ulfonylurea derivath’es (lilre tolbutamide, etc.) produce lusulin
amounts ; their diabetes is considered a “ Gcgenrcgulatipnsdialietes (IJcrtia
Bendfeldt and Otto, 1956). Those who are of the opinion that the adrenals
play a part in tliis “ Gegenregulation ” (Bastenie, in.-iG) fnid .support in the
observations of Szenas and Pattee (1950) that the glucocorticoid prof uction in
obesity is increased. This influence of overweight is also to be gathered from the
statistical data of Borth, Linder and Riondel (1957). Moreover the decreased
glucose tolerance which is so often found in obese subject.s is of the G.J.i.i.-
positive type, pointing to increased secretion of glucocorticoids too (.Arcndt and
IPSittiGG \9o0^

There exists a certain parallelism between the production of glucocorticoids
and adrenal oestrogens : operative stress or injection of ACTH increases the
urinar}^ excretion of both tj^ies of steroids (Decourt el ciL, 1951 ; Bulbrook c/ al.,
1958 ; Brown, Palconer and Strong, 1959) and cortisone therapy inhibits the
production not only of glucocorticoids but also of adrenal oestrogens (Smith and
Emerson, 1954 ; Block, McCarthy and Vial, 19596).

We are not of the opinion that every curve revealing decreased glucose toler-
ance is of necessity a reflection of an increased secretion of glucocorticoids (it
would have been preferable if also glucose-insulin tolerance tests of our ]iaticnts
could have been made). However, it seems reasonable to assume with French
gerontologists that at older age in the larger part of these curves the adrenal
cortex plays a part (Binet and Bouliere, 1955), and such an assumption already
satisfies the epidemiologist.

If our observations can be confirmed by others, a definite perspective regarding
mammary cancer is looming up. In the first place the indications in the treat-
ment of metastasized mammarj’^ carcinoma could get a more theoretical basis.
In judging the results of different hormonal kinds of therapy the tj-^pe of cancer
(“ ovarian ” or “ adrenal ”) could be included in the considerations. Our curve
of the “ ovamn ” type (Fig. 3) fits well in with the observations of Dao and
Huggins (1957) that after 54 years of age bilateral oophorectomj' is seldom
indicated. It could be found that young women with obesity and /or hypertension
are not helped sufficiently by castration alone but that in them at an early stage
the elimination of adrenal oestrogens must be advocated, either by surgicaf means

oy siippression of pituitary ACTH with corticosteroids. Finally the fact
could be understood why some patients who did not react favourably to surgical
1 o?o^ tn greatly helped by adrenalectomy (Block el al

bufohlm fact thTt /h response not being based on hormone independence
but on the fact that the ovanes were mactive in contrast to the adrenals

But of no less significance seems to us the possibility for more insight into some

pres "Sollri

mammarj. cancer. SceSg th/'emSmnSal"fert*^*l

that this carcinoma has a preferenerf^Tb^ V I rnust be stipulated

and that it is seen more LquentW t

and Africa (Segi et al, 1957 ; oltU and H.™giroi rgs*™ “ A’’™

446

DE WAARD, DE LAIVE AND BAANDEES-VAN HALEmjN

It is also well kiioira that the disease entities of the triad obesity essentiil
hjTjertension and diabetes mellitus (“ diahete gras”) are more freauent hi
inaterialty blessed peoples of Western countries than in these African and Asian
peoples (de Langen, WSS ; Smirk, 1949). Further, obesity, hypertension and
diabetes have also a hereditary aspect.

We suppose that the phenomenon of adrenal oestrus on account of its associa-
tion -ndth obesity, h 3 'pertension and decreased glucose tolerance (not only in
statistical biit probably also in pathophysiological respect) has also a hereditan-
and an environmental aspect, and that these aspects in turn determine tlie
hereditary and the environmental aspects of the “ adrenal ” type of mamiiiaiT
carcinoma.

With this hj'pothesis the peculiar feature shomi in Fig. 2 could be explained
wlty the bimodalitj’^ of the age-specific frequency curve of mammarj^ cancer is
not pronounced in the metropolitan areas of the U.S.A. in contrast to the
Scandinavian countries. In these parts of the U.S.A. the Western technical
civilization finds its summit, with all the life habits (nutrition inter alia) inherent
to it. If diabetes, obesity and hj'pertension and the phenomenon of adrenal
oestrus begin to occur a few 3 'ears earlier in life and if their frequencies increase
until age somewhat stronger than elsewhere in the Western world, the “ adrenal ”
group of mammar 3 ’^ cancers will overlap the “ ovarian ” group almost completely
masking the decrease of the latter group at menopausal age.

This environmental aspect of the older type of breast cancer may also explain
certain facts mentioned b 3 ’' McMahon (1957), himself a critic of the bimodal t 3 q)e
h 5 qiothesis, nameh’-, (1) the difference in mortality trends dining the 20th centur 3 '
between mammat^^ cancer among pre- and postmenopausal women respectively,
(2) the shift in the incidence break of the age specific morbidity curves of breast
cancer in Connecticut, (3) the differences between the curves of Danish urban and
rural areas.

In contrast to the life habits of Western countries we know of peoples with
way'^s of living in wliich diabetes, obesity and hypertension are relatively unknown.
Perhaps by'^ changing our nutritional habits we might be able to reduce not only
the incidence of these diseases but also that of adrenal oestrus and of the “ adrenal
tyqie of mammary carcinoma.

STJMJLARY

Morbidity and mortality statistics suggest that the population of mammary
cancer patients in Western coimtries is composed of two populations, each wit i
its own age distribution having their liighest frequencies about 48 years and
65 years of age respectively. Statistics from -the Netherlands show this bimodal

distribution too. ,, ,

In trying to find a possible basis for this phenomenon the h^othesis is dis-
cussed that the type of mammary cancer of reproductive age is caused by a i
ovarian dysfunction and the type of older age by an adrenal dysfunction. 1 iis
hypothesis is tested by applying epidemiologically

crine cytology that patients with obesity, essential hypertension and/or j

glucose tolerance often show signs of oestrogemc activity which are very probably

The results of this application seem to be in agreement with the mentioned

age DISTEIBUTION of MAMMAEV CAECIXOMA

.117

hjTiothesis. The i^ossible significance of
hereditary and the environmental aspects

these facts for more insiglit into tlie
of majnmary cancer is discussed.

The authors vish to thanlc tliose colleagues who helped to collect the material
for study • H. J. H. Bolhuis : K. Breur and 0. Karpialc-van J)ncl (Kadiothei.i-
peutisch Instituut, Rotterdam) ; Prof. Dr. J. F- 'isnho^r (Hcellcnndige Unn’ors.-
teitskliniek. Utrecht) ; D. Miete (Gemcente Zickenhms Arnhem) : J. II. D chtcn-
belt- A. L. E. Schaepkens van Riempst (Antonins Zickenhms, Utrecht); A.
Stofberg (Geertruiden Ziekenhuis, Deventer) and Dr. E. \ ei-schuyl J he cancer
morbidit}- figures were supplied by L. Jleiiisina, director of the Central Cancer
RegistrJ^ Dr. E. Tonkes and Jliss N. Vermeulcn gave their valuable assistance
in the investigation of the control group.

REFERENCES

A^-DEKSO^^ E., Reed, S. C., Huseby, R. A. and Oeiveu, C. P.— (1!).70) Cniircr, 3,
410.

Aeekdt, E. G. and Pattee, G. J. — (10.56) J. clin. Endocrin., 16, 367.

Basteme, P. — (19.56) ‘ Cortico.-surrenale ct diabctc humain ’. Paris (Masson).
Bektba:^, F. , Bendeeldt, E. and Otto, H. — (19.56) DIscJi. mod. Wichr.. 81, 274.
Binet, L. and Botjrliere, F. — (1955) ‘ Precis de Gerontologie ’. Ikiris (Masson),
p. 479.

Block, G. E., Burgess Vial, A., McCarthy, J. D., Porter, C. B. W. and Coller,
F. a. — ( 1959a) Surg. Gynec. Obstel., 108, 651.

Mem, McCarthy, J. D. and Burgess Vial, A. — (19596) Arch. Surg., 78, 732.

Boe, j., Hidiebfelt, S. and IVedervang, F. — (19.57) Ada med. sc/ind., Suppl. 321,
Table 50, p. 145.

Borth, R., Linder, A. and Riondel, A.— (19.57) Ada cmlocr.. Copenhagen, 25, 33.
Broito, j. B., Falconer, C. W. A. and Strong, J. A.— (1959) .J. Endocrin., 19. 52.
Bruinsma, a. H. and de Waard, F.— (1959) Ada endocr., Copenhagen, 32, 233.
Bulbkook, R. D., Greenwood, F. C., Hadfield, G. J. and Scowen, E. IC— (1958)
Brit. med . ./., ii, 7.

Clejuiesen-, j.— (1948) Brit. J. PMdiol., 21, 583.

Dao, T. L. and Huggins, C.— (1957) J. Amer. med. Hw., 165, 1793.

Decourt, J., Jayle, M. F., Crepy, 0. and .Judas, 0.— (1951) Ann. d’ Endocr., 12

Denoix, R F (1958) Proc. Znd int. Symp. on Mammary Cancer, Perugia, p 285
Desawe, P., Laiugne, J. and Adrianne, a.— (1958) Ibid., p. 37.

^CKE, K. H. Reiszig, G, — (1958) Arch. Geschwulstforsch. 12 379

Jacobsen, 0 —(1946) ‘ Heredity in breast cancer ’. London (Levis & Co.).

DE Langen, C. D. — (1958) Gene&sk. BL, 48, No. 3.

Lencioni, L. j. — (1953) J. clin. Endocrin., 13, 263.

McJIahon, B. — (1957) Cancer, 10, 1037

™ Ca,cr.

P..™rs, A. J. OvvauA, sA Wo,U m,h Or,.. 16, 267.

448

DE WAAED, DE LAWE AND BAANDERS-VAN HALEWIJN

VON PiKQUET, C. — (1930) ‘ Allergie des Lebensalters ; die bosartigen Gesclmilste
Leipzig (Thieme Verlag).

Segi, M., FuKTTSHurA, I., Fujisaku, S., Kuzihaea, M., Saito, S., Asano, K., Nagaika,
H., Noye, Y. and Kamoi, M. — (1957) J. nat. Cancer Inst., 18, 373.

Shtmkin, M. B. and Wyman, R. S. — (1945) Ibid., 6, 187.

Smith, 0. W. and Emerson, K. — (1954) Proc. Soc. exp. Biol. N.Y., 85, 264.

Sjiibk, F. H.— (1949) Brit. med. J., i, 791.

Stocks, P. — (1955) Schweiz. Z. Path., 18, 706. — (1959) Bull. World. HUli Org., 20,
697.

SzENAS, P. AND Pattee, C. J. — (1959) J. cUn. Endocrin., 19, 344.

DE Waard, F. and Baanders, E. a. — ^To be pubb'shed in Acta Endocr., Copenhagen.
World Health Oroanisation . — (1959) Tech. Pep. Ser. No. 168.

THE EKBQUENCY OF CANCER IN DIABETES 5IELB1TUS

G. HERDAX

From the Department of Public Health, UmvccsiUj of JiriMol
Received for imblication July 20, 1000

This paper is concerned rvitli the problem of whctlier the incidence of nmlig.iant
diseases in diabetics is significantly different from that in non-diabc ics. W Ink-
the material of autopsies from the Pathology Department of Bristol Unncr^I(\
and the clinical material from the Bristol Royal Infirmary confirm (he negative
association between the two diseases which has been the subject of previous
investigations, a critical examination reveals the new fact that such a negnth’c
association would seem to apply more to females. It is shown that the c.xplnnat ion
of a negative association between mahgnant tumours and diabetes is in agrccmcnl
with what has emerged so far about a hormonal implication via the disturbance
of the glucose metabolism in both diseases.

Section 1

The results of an examination of post mortem records in the Patliology Depart -
ment of the University of Bristol are shown in Table I.

Table I. — Mortality Due to Malignant Tumours and Diabetes from the Post
Mortem Records in the Pathology Department, University of Bristol

Total number of post-mortem records in the Bristol Pathologj- Department . 0317
Number of malignant tumours among them ...... 1212

Number of diabetics among the 6317 records . . . . . . 18!)

Number of coincidences of diabetes and cancer . . . . . . 19

Percentage of malignant cases in the total of 6317 cases . . . . Hl.o

Percentage of malignant tumours among non-diabetics (1193/6128) . . 19-.7

Percentage of malignant tumours among diabetics . . . . . 10- 1

Table II gives the distribution of post mortems (P.M.’s) according to age and
sex for the total of P.M.’s, the cases of malignant tumours, of diabetes, and the
cases of joint occurrence of mahgnant tumour and diabetes.

Particular attention is dravm to the fact that the age distribution (total) of
^abetics appears to be, by and large, a good rephea of that for diabetes morbidit\-
m the pneral population. It shows the characteristic increase in diabetes mor-

o0-80 a pronounced peak between 60 and 70 just
like the diabetes morbidity cunm in the general population. ^

On the face of it, the diabetics appear, according to Table I less suscentilile
to the mahgnant disease, the difference being about 9 per cent There Jc nJri ■

111 the age distributions as displaved in Table TT 1 1 nothing

onJTSffe^lr 7

450

G. HERDAif

Table II.-~^gre and Sex Distribution of (he Post MoHem Cases
Summarized in Table I

Age

Total of

P.M.’s

Cases of
malignant
tumours

Cases of
diabetes

Malignant
tumour plus
diabetes

group

Total

jM.

Total

T^i

M. F. To

0-9

. 840

057

1497

32 .

10-19

. 99

190

15 .

20-29

. 149

151

300 .

21 .

30-39

. ISO

185

371 .

41 .

• —

40-49

. 307

281

048 .

124 .

1 2

50-50

. 037

309

1006 .

160

114

280 .

36 ,

3 i

00-09

. 717

450

1107 .

224

119

243 .

62 .

2 3

70-79

. 503

351

854 .

170

275 .

4 2

80-89

. 133

120

253 .

75 ,

11 .

90-99

24 .

4 .

100 -1-

—

1 .

—

— .

— —

Totals

. 3041

2670

0317 .

753

459

1212 .

128

18D .

11 8 1

may first regard tlie percentage of malignant tumours among the total of autop-
sies — 6317 — as the statistical population percentage of malignant disease, and the
number of diabetics — 189— as a random sample of the total. We then have,
for the standard error of the population percentage in such a sample of 189
specimens

/1 9-2 X 80-8
V 189

2 - 86 %

and 3cr = 8*56 per cent added and subtracted from the basic percentage, 19*2,
gives 27-76 per cent and 10-64 per cent respective!}' as the upper and lower limits
Avithin wdiich we could expect the percentage of mahgnant tumours in a sample of
189 specimens to lie. The observed percentage of 10-1 among the 189 diabetics
lies beyond the 3o- limit and is, therefore, to be regarded as highly significant of an
assignable cause.

The same answer is obtained if we estimate the population percentage on the
basis of the sample percentage, by means of the chart on page 61 of ‘ Statistics of
Tlierapeutic Trials ’ (Herdan, 1955). Entering the chart AA’ith p = 10-1 on the
upper horizontal scale and projecting the crossing point of the vertical with the
curve # — 189 to the left (or right), we read there the difference beriveen the
upper limit for the population probability p„, and p as slightly less than 8, and
obtain, therefore, the upper limit p„ as slightly less than IS, which is significant)}
smaller than the observed figure, 19-1 per cent.

A second possibility is to regard both the percentage of malignant tumouzo
among the total of P.M.’s and that of coincidence cases of the two diseases as
samples from the statistical population of P.M.’s in the wider sense, ^y in t zc
AAdiole of the country, and determine the significance beWeen the two. The resu
is essentially as before.

Section 2

It is noAV important that there is a remarkable stability of the figures
for the malignant disease, in general, and among diabetics, not on y ‘

country, but between countries, and significance tests could also be cam

CAXCER

AXD DIAIIETES MEI^EITUS

.};-)!

„„ the percentages et the same

the^SSS,!isS

Table III ^vhich gives relevant numbers as m Table I for six uuesug*

the
Table
that kind.

Table 111.— MorfaUfy Dnc to Malujmmt Tumour.'^ ami Diabric.s from
German Post Mortem Records

and

Post mortems . . . •

Malignant tumours

Diabetics . ... ■

Malignant tumours and diabetics .

Diabetics as percentage of total
P.M.’s.

Malignant tumours as percentage of
total P.M.’s.

Malignant tumours as percentage of
non-diabetics

Malignant tumours as percentage of
diabetics

Time of publication

Ivruger Mnllory Eskuclicu
(1<H0) In. (104")

5.844 — 10.04S

731 — 1.804

122 307 23f>

10 24 21

2-00 — 2-4

Wolscli

(10.52)

13..54(>

2.271

402

2-07

Eii-lilt-r

(10.54)

10.242

3..524

037

3-3

Wi-nicr

(10.53)

2.5.147

4.080

70.5

2-S

.Si-liriiU-r

(1000)

22.071

4.142

351

1-.53

12-15 — 1" 3

\e>-i~

18-3

10 -.8

18-1

12-7 — 1'-"

17-3

18-0.5

20-4

18-3

8-2 S-4 8-3

o-os

0-27

7-1

8.8

1940 1044

1052

10.54

1055

1050

We shall consider as a particular case the last of these investigations tvhich
does not differ essentially from the others in its results. On the basis of 22,5)71
P.M. records from the University Clinic of Halle a/S the percentage of malignant
cases resulted as 18, remarkably close to the figure for Bristol, 1 5)'2. The percent-
age of coincidence cases of diabetes and malignant disease tvas for Halle S-S per
cent, again remarkably close to the figure for Bristol, 10-1 per cent. The signi-
ficance test shows that we may regard the basis of the Uvo investigations as one
statistical population of P.M.’s in which the percentages of tumours, 18 and lfi-2
differ by not more than could be accounted for by random sampling from that
one population. The same applies to the percentages of coincidence cases, 8-8
and lOT per cent.

To counter the possible objection that the negative association between malig-
nant disease and diabetes is only apparent and due to the fact that the diabetic
does not live long enough to be attacked by malignant disease, we give below the
age distribution of the coincidence cases which shows the coincidence cases as
not being different in age distribution from the total of cancer cases.

The same is, by and large, true for the German coincidence cases. Both
malignancy and diabetes and also the coincidence cases, increase witli age and
hen decrease again. The peak of the age cunm in Germany lies betn-een 55 and
o years for malignancy and for the diabetics betnTen 61 and 70 years and for
the coincidence cases between 71 and 80 years This means tbuf Jrl! ’r I /
reach a sufficiently high age to be evnosed to tb« ^ today diabetics

like other parts of the popltion "'“''e-"!. iisMse

452

». HEEDAN

Table IY,-Age, Sex and Site for the 19 Coincidence Cases of Cancer and
Diabetes Mentioned in Table I

Site

Breast

Gall bladder
Kidney
Larynx
Lung

Pancreas .

Eectum

Stomaeli .

Leukaemia

Lymphocarcinoma

Sex

Age

31.

Altliougli the difference between the Bristol percentages and those of Halle
of the same denomination are not statistically significant, yet one might be
somewliat uneasj’’ about what looks like a slight s^^steraatic increase in the Bristol
percentages : 1 9- 2 against 18 per cent for malignant cases, and lOT against S-S
per cent for coincidence cases. However, the explanation may lie in the change
of diabetes incidence with time. As is well known, diabetes is, in general, on
the increase in both England and Germany and so is cancer of certain sites. In
spite of the agreement between the Bristol figures and those from Halle a/S
there is a difference in the sex distribution between the two samples. For Halle
the relation between M. and F. among the diabetics is 0-78 to 1 and that for the
coincidence cases 0-82 to 1. For the malignant tumours without diabetes the
relation is 1-33 to i. The corresponding figures for Bristol are 0-48 : 1, l-SS : 1
and 2-24 : 1. The statistical influence of the striking difference in sex distribution
for tumours and diabetes upon the coincidence rate is discussed in Section 5.

Regarding the relation of the analysis of post mortem records to mortality
statistics of the -whole population in England and Wales, the following figvires
■ff'ere obtained for 1950 :

Deaths as percentage
of all deaths

Cancer

(%)

31.

0-170

0-172

17 approx.

Diabetes

31.

0-0046S

0-5

0-0098D

1-0

Although the percentage of cancer cases among the P.M.’s is in fairly good a^ee-
ment ivith that in the total population— 19 as against 17 per cent—the pe^entage
of diabetics among the P.M.’s, approximately 3 per cent, is rather higher tlian _
percentages in the total population, 0-5 for males and 1-0 per cent for lemale...

CAKCER AN» DIARETES MEI-EITKS

4r,:5

which .night conceivably have been ' Mnw.ner,

incidence casea, f So.f ordW.'dIe il. ^.d i-,in .be

populatto’is a“most precisely that for the dinbeties among .be I'.JI. a. We hate

61 H. : 128 F. := 1 : 2

and in the population, u’e have the diabetes percentage of all deaths

0-.5 M. ; I-O F. = 1 ; 2.

Section i .

The stability of the corresponding percentages in different investigations is so
remarkable that it calls for comment from both the medical and statistical '"'h' f‘-
It is often maintained that P.M/s cannot be regarded as a random sainple of the
mortality in a given countrj', since there is no definite rule as to wbicli eases are
to be subjected to autopsy. It largely depends on the preference "'liicb the
consultants in question have in this respect, and this may be different at difrerent
times according to %vl\icli tj’pe of disease is specially in the foreground of niedic<i
interest. This is then adduced as a reason for the doubtfulness of comparability
between mass results based on P.JI.’s.

While it is readily admitted that the basis of true comparability of statistical
results is that they were obtained in the same manner, yet to overdo tlii.s and
restrict the possibility of comparison in this way would be against tlic very nature
of statistics. Statistics is a method to be applied where the strict rcipiircments
of laboratorj^ experimentation cannot be obtained. In observation of masses
of events wlricb is the verj.- essence of statistics, it is just the impossibility of
following each indi^^dual case which makes statistical methods ncces.sary. 'I'liaf
in spite of the lack of complete comparability between mass results there should
be a certain stability is the verj" basis for the conception of medical statistics ns
conceived by Dr. Farr.

Appljdng these considerations to P.il.’s, we must, on the basis of the jiresent
investigation, arrive at the conclusion that in spite of the P.lM.’s at different hospi-
tals being carried out by different persons with different points of view, a suffi-
ciently great number of these autopsies — ^provided only that there is no true, i.e.
intentional, bias of the investigator — can he regarded as a sample of the population
of P.il.’s. Rightly understood, a conclusion of this kind expresses in words
what must be the basis of any medical inference about a certain amount of pro-
tection from malignant diseases in diabetics. As a biological or pathological
fa,ct, it must apply to mankind in general, provided only that the living con-
ditions, etc., are comparable. *

However, in spite of giving, by and large, a fair picture of the relations of the
rates in the population for the two diseases in question, we cannot, in the strict
sense of the term regard a given mass of P.M.’s as a random sample of the popula-

of proportions we have noticed amo^g^ the
c ifferent raassas 0/ PJI. s justifies regarding these masses as representinri a statisti-
cal popnlalion in its own right. What it loses by not being striX a random

464

G. HERDAN

not matter for our argument, since the percentages which we compare, i.e. those of
malignant tumours and diabetes, are both from the same population of P.JI.’s
The difference from the general population influences therefore both percentages
in the same way. °

It should also be noted that we are not concerned with the percentat^e of
diabetics among the P.M.’s, which may, and probably will, differ from that in the
general population, but onty with the percentage of diabetics among tumours,
i.e. with the coincidence cases. Even if the number of diabetics were system-
atically too small compared with what it is in the general population, this need
have no influence upon the number of coincidences.

Section 4

According to the literature, the clinical experience did not always seem to
bear out the conclusion reached from a study of P.M.’s. Thus, Constam (1950),
who gives the percentage of the incidence of malignant tumours in non-diabetics
as 3-0 per cent and among diabetics as 5-1 per cent.

In order to check these findings I obtained from Bristol Royal Hospital,
Royal Infirmary Branch, the corresponding data for 7 3 mars, 1953-59, which are
set out in Table V.

Table V . — Morbidity Due to Malignant Diseases and Diabetes in the
Bristol Royal Infirmary

Mnlignnnt diseases

registered 4

Year

Total number
of in-patients

Number

percentage of
in-patients

Total number
of patients
Avith diabetes

1953

8.587

463

5-39

142

1954

9,150

409

4-47

145

1955

9,592

405

4-22

169

1956

9,051

753

7-80

171

1957

9,446

578

6-12

202

1958

9,524

587

61G

205

1959

8,755

488

5-57

195

Totals

04,705

3,683

5-69

1,229

Patients with joint
occvirrence of diabetes
and malignant tumour

t — •* 1 ,

Percentage of
Number diabetics

3 2-U

6 4-14

5 2-92

2 M7

S 3-9G

4 1-95

7 3-5S

35 2-84

The results are most interesting in so far as thej’^ seem fulty to support the
conclusion obtained from our P.M. data. The incidence of malignant tumours
among diabetics is considerabl}’’ less than among all in-patients, the ratio be ateen
the two overall percentages, 2-84 and 5-69, being approximately 0-50 winch is in
good agreement with the ratio of the corresponding percentages ob ame roi
our P.M. data, 10-1 and 19-2, which is 0-53. The percentage of tumours amoi „

non-diabetics is 5-75.

On the basis of our data, be they from P.M.’s or from clinical e-'^penence, a
have therefore, no reason to doubt the significantly reduced incidence of maiignam
tumours among diabetics.

Section 5 . , xi „

Inspection of Table II with regard to the se.x distribution, revea s ^ m
able fact that the number of female diabetics is more than tA ic

CAXCKP. AXn DIAUKTKS .Mi:i,I,ITrs

■jr.r,

connection witli llic smaller fre<|neiu\v of mnlipnanl (liscascs

1 ./I 4 . .> 4 K ^4 4 1 .

1-64 ; 1 ). at once snggests Hint the negative ns'-'oeiation
niav not lie (|uifc' lioinogeneons as

diabetics. This, in

in females (M.; F. 1 VPT . 1 iVV .'ll

between diabetes and malignant, tumours may
regards the sexes. Using the following .symbols

C'j, = Total of inalianant tumour patients
T = Total of P.M.^s

Cjy = Malignant tumours among diabetics
D ~ Number of diabcfic.s

and the subscript
we have for males

for male and , for female

and substituting our figures

and for females

CJT,„=A\-

'l>m

0-207= yf, X o-is’f)
0-207

0-1 SO

r,/?’, = K, ‘i'!i

in figures: 0-172 = /i', X 0-003

iu = 0-172/0-003 = 2-73

autons^Mic: V'® incidence of malignant tumours in the population of

figlrS-ls or r"' t lie Corresponding

nSles is 87 per cero?t?at'ln of malignant tumours among iliabotic

it is only 37 per Siit autopsies population, whcrea.s for females

we Sd SrAVe'fbsm^d'^v^^^^ T taken into account

is, by and large trve otdvZ7hl f''''^T''^ as.socation between the two diseases
nant diseases^in the total nf f®-'- For males, the incidence of maiig-

-ongdiahSiraufop.?es a^ " -sig^n-ficantly different from tiu^t

Section 6.

diaSTs'the tranSoi^'T?^ P®*'*^'’*ogicaI change in gli

g^issiiiiiss

standablC they U'w'Tl"

which would^K r age does not snfF r ' under-

8i«=ose r^UiwTr'’’’’ orSi™

allowed to accumulate in the oil] but i?„Sd The

used on a large scale for the

I gIuco.se metabolism. In

456

G. HERDAN

production of the energy by glycolysis because the other pathway, respiration
IS much reduced in the tumour cell. ^ ’

The pioneer Avork of Warburg on the energy-producing reactions of tumours
lias Jed to a vast amount of research on the subject. As a result, it is now clear
tJiat these reactions depend to a large extent on the metabolism of carbohj^dr<ates,
and that the metabolic pathways of neoplastic cells differ considerably from those
of normal cells. Tumour cells can carry out both aerobic and anerobic glycolysis
but the latter process is particularly enhanced and very high rates may be manifest.

Whilst the shift of emphasis in the glycolitic and respiratory capacities of
neoplastic cells from that obtaining in normal cells is well recognized, its signi-
ficance has been a point of controversy for many years. Warburg (1956) considers
it to be an irreversible alteration resulting from a change in the mitochondrial
respiratory sj’’steni. In Ins view, damage to the respiratory apparatus in mito-
chondria of normal cells is followed by a selective process favouring the surAuval
of cells capable of increased permeation as a means of compensation. Many
other investigators believe however that the process of carcinogenesis depends
hasicalty on nuclear changes, and Ifit and Griffin (1958) regard primarj’’ damage to
respiratorj’^ grana as being significant onlyr if it results in nuclear alteration.

Thus in neoplasms there is a disturbance of carbohydrate metabolism localized
in the tumour cells, v'hereas in diabetes mellitus a generalized alteration of the
very same metabolic process exists. Henderson and LePage (1959) pointed out
that in the presence of an unusual carbohydrate metabolism certain compounds
may become essential to the groAAdh of a tumour Avhich wmuld otherwise be
regarded as non-essential. In their review they summarize experimental obser-
vations which could be interpreted as erridence of an avidity of tumour cells for
glucose such that the normal tissues of the host are depleted of sugar. Prom this
it seems that a neoplasm would be capable of influencing the severity of an existing
diabetic state, so that the local disturbance of carbohydrate metabolism in the
tumour affects the general state of carbohydrate metabolism in diabetes.

This is the reverse of the possible influence of diabetes upon the incidence of
human tumours AAdiich emerges from tlie P.M. analysis given in Sections 1 and 2.

Acknowledgments are due to Mr. A. E. J. Turner, Group Medical Records
Officer, United Bristol Hospitals for providing tlie information used in Section 4
of tills paper.

REFERENCES

CoNSTAM, G. R.— (1950) ‘Therapie des Diabetes mellitus’. Basel (Verlag Benno

Scinvabe & Co.). , , , -n- i ’

Eichlee, R.— (1954) ‘ Uber die Haufigkeit u. Lokahsation d. ICrebses b. Diab. niellit.

Inmig. Diss., Leipzig.

Eskuchen, G. Th. — ( 1947 ) Em., Hamburg.

Henderson, J. F. and LePage, G. A. — (1959) Cmcer Res., 19, 88/. ..p,, • p ui

Hbkdan, G.— (1955) ‘Statistics of Therapeutic Trials . Amsterdam (Elseiier i

Company).

Kit, S. and Geifein, A. C.— (1958) Cancer Res., 18, 621.

KEiiGER, B.— (1940) E. /Uefo/o?-sc/i., 50, S.126 in? SQ7 ff

Rockstroh, H. and Schhotee. H.-(1960) jShmeh. med. Wschr., 102, 89/ ft.

Waebueg, 0. — (1956) Sc/eace, 123, 309. ^ Ar„.EiirD-

Welsch, H.— (1952) ‘ Karzinom u. Diabetes . Jnatig. Diss., Marburg.

Werner, W.— (1953) Arch. Oeschwulstjorsch., 5, 334.

■n'A'RT.Y fPROPHYLACTIC) OOPIIO]?lCCT():\lY AND
ADRENALECTOMY IN CARClNOi\IA OF THE RHEAS T
AN INTERIM REPORT

D. H. PATEV

From the Department of Surgical SIiuUcm, Mi,hnrs-r.r llospilni. homhn. MM

Received for iniblieiitioii Mny 5, RlliO

Early in 1953 following the pioneer work of Huggins, we hegan to (real,
selected cases of advanced carcinoma of the breast, by bilateral ()opIu)rec)oiny anrl
adrenalectomjL AVe confirmed, as have many others, tlmt m a i)ro])ort ion ol
cases palliation inav be acliieved, and in ci very small ])ro])ortion pos.siblc jno-
longation of life, the longest survival time in onr series being -1 years. In most,
cases however, times both of palliation and of survival had to be measured in
months rather than years. We therefore considered the ])ossibility that, the
limited benefits of hormonal surgery might be related to the advanced stage of the
disease at the time of operation, and might be inpwoved by earlier operation.
^Ye decided to put this lnT)Othesis to the test by operating on a small number of
patients with a bad prognosis as soon after the primary operation ns ])racticable.
The first patient was operated on in November 1954, and since that date there
have been 10 further cases. Many years would have to ])as.s before there could
be positive evidence that the operation was of value, but evidence against the
operation being of value could accumulate in a shorter time. Such cvi<lence
appears to be accumulating, and is reported in the present ])a))er.

SELECTION OF CASES (Table I)

We have deliberately kept the numbers small. Two essential criteria of
selection have been adopted. First, the prognosis must be bad, and secondlv,
the patients must be able to appreciate the nature of the problem, though wc
naturally dwelt on the hopeful aspect of Avhat Ave Avere attempting. Li all cases
the husbands AA^ere also interAueAved and the problem explained. In general, tlie
policy Avas to perform bilateral oophorectomy and adrenalectomy Avithin a feu-
months of the standard breast removal and axillarj’- elearance, and when the
patients Avere apparently free of disease. Exceptions Avere Cases 1 and 4 in Avliich
there Avere supraclaA-ieular glands, Case 2 in Avhich there Avas a parasternal recur-
rence Case 3 in Avhich there Avas evidence of a paratracheal gland on X-ray of
the chest, and Case 5 in Avhich there Avas a minute local skin nodule But in all
these five cases the amount of groAvth present was minute in comparison Avith the
cases ,vith wide dissemination normally treated by hormonal surgm (D e ma^^n

“ “topical examinaSof

patMogica. oHterion ,vas preaeat i/s of

45S

D, H. PATEY

Attempted prophjdactic operations of tliis type must also satisfy the Wo humane
cntma that tliore is substantially no operative mortality and no appreciable
morbidity. In this seri^, there were no operative deaths ; all patients have been
seen evey month with Dr. John Nabarro at a special adrenalectomy clinic vhen

their replacement therapy was regulated, but apart from this have been leading
normal healthy lives. icaum,.

Table I

Date of
ndrennlectoiny

Time since

Patient

Age

Indication for
operation

adrenalectomy

(months)

Result

1. Jlrs. K — .

. Nov. 1054 .

Supra-clavicular glands

Died, re-

2. Mrs. D— .

. Aug. 1955

Para-stemal recurrence

currence.

Ditto.

3. Mrs. 0-- .

. Sept. 1955 .

Invasion nxilJnrj' int.

Alive and

■1. Mrs. E — .

mammarj’ and para-
tracheal glands

well.

. Feb. 195G .

Acute pregnancy care.,

Died, re-

5. Mrs. IV— .

invaded supra-clnvi-
cular glands

currence.

. Nov. 1957 .

Invasion axillarj’ and

Alive, re-

G. JIrs. T-- .

int. mammary glands
and minute local no-
dule

currence.

. Dee. 1957 .

Invasion axillary and

Died, re-

7. Mrs. L — ,

int. mammary glands

currence.

. Jan. 1C58

Ditto.

Alive and

well.

S. Mrs. H— .

. July 1958 .

Ditto.

9. JIrs. M— .

. Got. 1958 .

»♦

Alive, re-

currence

10. Mrs.G— .

. Jan. 1959

Alive and

well.

H. Mrs. P— .

. Oct. 1959

♦ »

. 7

Ditto.

RESULTS (Table I)

TJie patients are tabulated in clironological order of operation, and it will be
seen that of the first 6, i.e. those with the longest follow-up, 4 have died of the
disease, one is alive with recurrence, and one is alive and well just over 41- years
after the adrenalectomy. Apart from this last case, 4 other patients are alive
and iveU, but the longest time since operation is only just over 2 years, and one
patient operated on as recently as October 1958 though alive has a recurrence.

DISCUSSION

The fact that, though the follow-up is only a short one, 6 out of the 11 patients
are either dead from recurrence, or though alive have recurrence, is eiddence
against the value of early oophorectomy and adrenalectomy in carcinoma of the
breast. The evidence is not conclusive and may not be so for some years, since t le
patients that have died or developed recurrence may have had “ hormone-indepen
ant ” tumours, and some of those still alive and weU may have hormone
dependant ” tumours and rvill continue to do well. Case 3 might well belong o
the postulated latter group since not only has she remained well for 40 jmars, u
the paratracheal gland noted on X-ray examination of the chest has remaine
unchanged during this time. Against this more optimistic view however is

TROPHYLACTIC OOl’HOBKCTOMV AND ADRICXAIAR’TDM Y

fact tliat Anth the excejition of Case 3, all the other o patients followed jip for 3
years or more fall into the dead or recurrent gronj), and “ honnone-de)>endanl- ”
tumours are generally thought to constitute a higlicr pro])ortion t lian I in (1. But.
in addition to this, there was in fact evklcucc of “ hormonc-deiiendaney " in some
of these patients. Thus in Case 2, the ])araslenial recurrence (lisa])i)eared follow-
ing the hormonal surgery, and in Case 4 the supraclavicular glands, in hoth rases
these particular manifestations not rea])pcariug during the terminal generalized
phase of the disease. Thus, though one cannot yet he detinit e, one must, at ]iresent
unfortunately conclude that early oojrhorectoiny and adrenalectomy are unlikely t(j
offer a significant contribution to the problem of carcinoma of the breast .

Sb'MMAKY

1. In 11 patients rvith carcinoma of the breast, oo]diorectomy and adrenalec-
toinj’ have been performed as soon as practicable after the primary removal of the
growth bj’’ standard surgical methods.

2. Though the follow-up is only a sliort one, G patients cither have alreadv
died of the disease, or though alive have developed recurrence.

3. A longer follow-up Avill be needed before final conclusions arc reached, but.
the interim conclusion seems justified that early oophorectomy and adrcnnlccton)\'
are unlikely to offer a significant contribution to the problem' of carcinoma of the
breast.

I would like to acknondedge my indebtedness to Dr. John Nabarro whose
close collaboration in the selection of patients for operation, the operative aiid
post-operative management, and the follow-up, have been an esaentml and
stimulating part of this study.

REFERENCE

Hakdley, R. S.— (1958) Quoted by Patey, D. H. ‘Endocrine
Cancer ’. Ed. A. R. Citbeie. Edinburgh (Livingston), p. 25

Aspects

of Breast

460

17-KETOSTEROIDS AKD THEIR FRACTION'S IN W 01 \rR\*
WITH BREAST CANCER TREATED BY ENBDOCRINE S^GeS^

A. W. SIM, R. HOBKIRK, HELEN J. STEWART
D. W. BLAIR Ain> A. P. M. EORREST

From the University Department of Surgery, Western Infirmary, Glasgow
Beceived for publication June 9, 1060

Thb neutral 1 7-ketosteroids excreted in the urine of normal women are mainlj^
metabolites of the hormones of the adrenal cortex. They can be separated by
chromatograpliy into two main fractions, those which have an oxygen atom at the
11-position in the steroid nucleus (the 11-oxy 1 7-ketosteroids) and those in which
this function is not present (the 11-deoxy 17-ketosteroids). The 11 -oxygenated
steroids are mainljr derived from the 21 carbon atom (C21) glucocorticoids, i.e.,
cortisol Avhile the 11-deoxy compounds are for the most part excretion products
of the Cl 9 androgenic liormones. In this paper we report the results of esti-
mating these urinarj" steroids in women -noth advanced breast cancer treated by
endocrine surgery.

METHODS

Urinarj'- 17-ketosterolds and their fi’actions were estimated in women with
metastatic or recurrent mammary cancer treated by oophorectomj^ adrenalectomy
or radioactive implantation of the pituitary gland (Forrest, et al., 1959). Collec-
tions of urine for assaj'^ were made wJiile the patients were in hospital, the urine
being collected in clean bottles and stored below 4° C.

Total neutral 17-ketosteroids were estimated in duplicate on 100 ml. ahquots of
total 24-hour urine collections by the method advised bj^ the Clinical Endocrino-
logical Committee of the Medical Research Council (1951). After acid hjArcIj^sis
the free steroids were extracted vdth carbon tetracldoride and phenolic steroids
(oestrogens) removed by an alkaline rvash. The concentration of remaining
neutral 17-ketosteroids rvas estimated colorhnetrically by the CaUow-Zimmerinan
reaction.

17 -Ketosteroid fractions . — ^Fractionation of 17-ketosteroids was carried out
on 24-hour or pooled 72-hour collections of urine by the method described in
detail elsewhere (Hobkirk, 1958). After initial saturation of the urine with
ammonium sulphate ((NH4)2 SO4) the glucuronide and sulphate steroid conju-
gates were extracted bj'^shalcingwith ether-ethanol (3: l)and successively hydroly-
sed by limpet jd-glucuronidase and hydrochloric acid at room temperature. The
resulting free steroids were extracted with ether, washed with alkali and ketonic
fractions prepared udth Girard T reagent. These were separated into 3a and 3p
hydroxysteroids by treatment with digitonin. Further fractionation of the 3a
hydroxysteroids into ll/?-hydroxyaetiocholanolone plus 1 l^-hydrox3mndosterone,
11-ketoaetiochololone, ll-ketoandrosterone, androsterone and aetiocholanolone
was carried out by chromatography on paper in a ligroin-propylene glycol system.

4(51

UKIXAHY 1 7-KETOSTEUOTWS IK EEEAST CAKCEH

Quantitative estimations of each fraction was carried out hy
the Callow-Zimmerman reaction (Wilson, 1954), ahsoluie values or each stem, 1
being obtained by reference to calibration curves for the pure comjioumls \\ In rc
amoLts permitted, ultraviolet absorption curves of the cln-onia ographic
in concentrated sulphuric acid yielded further proof of identit.y. 1 he inobablt.
source of these fractions are shown in Table I.

Table I . — Neiiiral ll-heloslcroid Fracliom in Female Urine
and {heir Probable Source

Hormone

Cortisol

ll^-Hydroxy A*-Androstendione
Cortisone

Urinnr}' melnbolite
1 1 ^-Hydroxynndrostcrone
1 1 /?-Hydroxynetiocholnnolone
1 1 -Kc tonndrosterone
1 1-Kctonetiocliolnnolonc

Kotosloroid frnctinn

1-oxy I7-k('tostoroids

3 Hyilroxyl
orientiilion

rriiclion

Androsterone

A*-Androstene 3, 17-dione - Aetiocholnnolonc

^11-dcoxy 17-ketosteroids

Dehydroepiandrosterone

Dehydroepinndrostcrone

3/? frnrtioii

RESULTS

Tolal neutral ll-hetosteroids

Adrenalectomy and oophorectomy . — ^In twelve post-menopausal women with
breast cancer total neutral 17-ketostero5ds were estimated in at least tlirco 24-
hour collections of urine before and in at least three collections made 1 to 3 months
after adrenalectomy and oophorectomy. Post-operative urine was collected u’hilo
patients were receiving maintenance cortisone (50 mg. /day) and also during a
5-day period when they u'ere given 120 units corticotrophin (ACTH) gel daily by
intramuscular injection. Six of the twelve patients had objective evidence of
regression of cancer following the operation ; in the others the course of the disease
was not affected.

The excretion of neutral 17-ketosteroids in the urine was significantly depressed
by surgical removal of the adrenals and ovaries (Table II — mean difference -l
standard error: 1-4 ± 0-6 mg./24 hr; P < 0-05). The mean fall in the shi
patients whose cancer regressed was identical to that in those who did not benefit
In neither group of patients did ACTH produce a significant increase in keto-

steroid excretion (Table II — mean difference in responding patients 0'4 ~ 0-G

mg-/day ; in non-responding patients -f 0-2 ± 0-5 mg./day. Pig. l).

Table II. — Mean Excretion of Total
12 Patients with Breast Cancer Before

Neutral 11 -ketosteroids {mg. per day) in
and after Adrenalectomy and Oophorectomy

Before ndrenalectomj-— no cortisone ,

.After adrenalectomy— cortisone 50 mg. per day
After adrenalectomy— cortisone 50 mg. ACTH 120 units

Cancer

Cancer not

regre.ssed

improved

(6 patients)

(0 patients)

Total

4-5

4-3

4-4

2-9

3-0

2* 7

2-9

462

SIM, HOBKIRK, STEWART, BLAIR AND FORREST

and after implantation of the pituitaiy with Yttrium-90, Urine was collected
post-operatively while patients were receiving maintenance cortisone 50 mg /day
The pre-operative results are the means of three to six determinations 'while
the post-operative means are those of all estimations carried out within 3 months of
implantation. Nine patients had regression of cancer as a result of pituitary
destruction. Neither in them nor in the other nine patients whose cancer failed

(Maintenance Cortisone 50-75 mg.)

Fig. 1. — Effect of ACTH (120 units per day for 5 days) on urinary neutral 17-ketosteroids follovring
adrenalectomy and oophorectomy in twelve patients irith advanced breast cancer.

to respond, did pituitary implantation lead to significant reduction of total
1 7-ketosteroid excretion (Table III ; Fig. 2).

Table III.— Jf can Excretion of Total Neutral ll-hetosteroids [rng. per day) in
18 Patients Before and After Implantation of the Pituitary with Yttrhim-^H

Cancer
regressed
(!) patients)

3-5

-0-3±0-4
>0-4

Before implant — no cortisone

After implant — cortisone 50 mg. per day

Mean difference ....

Cancer not
improved
(9 patients)

Total

3-6

3-5

3-9

3-5

-f-0-3±0-9 .

—

>0-7

fractions ivere

estimated

Neutral 1 7 -ketosieroid fractions

Pre-operative estimations . — ^Neutral I7-ketoster(
on 3-day pools of urine in twenty-Uvo patients rvith uuvnuucu „

treatment by endocrine surgery. Five of the patients had functioning
at the time of estimation (marked ^nth P. Fig. 3). In two patients only ^
11-deoxy and 11-oxy fractions were estimated ; in the other tventj p

URINARY 17-KETOSTEROIDS IN RREAST CANCER

fractionation was carried out. Tlie excretion of tliesc fractions in twelve jiatienls
who had subsequent regression of cancer was quantitatively similar to that, in
ten patients who had no response to treatment (Table IV ; Fig. .‘I). The means of

>•

•T3

^ 4

F 3
2
I

CLINICAL IMPROVEMENT

CLINICAL DETERIORATION

BEFORE IMPLANT

AFTER IMPLANT
Cortisone

50 mg per day

BEFORE IMPLANT

AFTER IMPLANT
Cortisone
50mg per doy

<-o

3 >2 ay.

M£AN.3.ea9

UCAN.3‘9 ay

a 1 ® eiglitcen pnticnl.s witli ntivnnc

before and after implantation of the pituitary with Yttrium.no.

464

sm, HOBKIRK, STEWART, BLAIR AND FORREST

Table n —ilfeaji Excrehon of Urinary/ ll-ketosteroid Fractions in 20 Women
with Bt east Cancer Before Endocrine Surgery. Two Patients in Whom Onlu
ll-(^eo:K!/ 11-ketosteroids were Estimated are Not Included in
this Table but Are Included in Tables V and VI and Fig. 3

ll-oxy n-ketosteroids ll-deoxy ] 7-ketosteroids

Response to

ll-OH E

fraction

-A

endocrine surgery

n-OH A

11 -KE

11-KA

Total

Cancer regressed
(11 patients)

0-52

0-31

0-10

0-93 .

0-93

0-71

0-12

1-76

Cancer not improved
(9 patients)

0-74

0-44

0-08

1-26 .

1-32

0-87

0-03

2-22

11-OHE = n/?-Hydroxyaetiocholanolone.
IJ-OH A = Il^-Hydroxyandroaterone.
11-KE = II-Ketoaetiocholanolone.

11 -K A = 11-Ketoandrosterone.

E = Aetioeholonolone,

A = Androsterone.

Table V. — Comjiarison of the Mean Pre-operative Excretion of ll-ketosteroid
Fractions in Twelve Patients Whose Cancer Subsequently Improved Folloiving
Endocrine Surgery and in 10 Patients in Whom There Was No Response

Response to
endocrine surgery
Cancer regressed .
Cancer not improved

Number of
patients
12
10

U-oxy 17-ketostero!ds

^ ,

Arithmetic Logarithmic

0- 94 0-864

1- 23 1-038

ll-deoxy 17-ketosteroids

t — - ^

Arithmetic Logarithmic

1- 83 1-073

2- 28 1-236

Mean difference ± S.E. .
P ... .

0-29±0-258

> 0-2

0-174±0-130

> 0-1

0-45±0-617

>0-4

0-163±0-198

>0-4

Post-operative estimations . — Estimations of neutral 17-ketosteroid fractions
on 24-hour or pooled S-dajr collections of urine tvere made in sixteen patients after
adrenalectomy and oophorectomy and on pooled 3-day collections in twenty-
two patients after Yttrium-90 implantation of the pituitaiyL In both groups of
patients urine was collected within 12 months of surgery u'hile cortisone therapy
(50 mg. /day) was being taken. Significant amounts of ll-oxy 1 7-ketosteroids
were present in the urine of all patients, being the excretion products of mainten-
ance cortisone (Fig. 4 and 5). The amounts of these fractions were not related to
the clinical response (Tables VII and VIII). The means in the groups of patients
were statistically compared as above ; no significant difference Avas demonstrated
(Table IX).

Adrenalectomj'^ and oophorectomy led to the complete disappearance of ll-
deoxy 1 7-ketosteroids in four of ten patients Avhose cancer responded to the opera-
tion and in five of six patients whose cancer did not improve. In six patients
in the responding group trace amounts of aetiooholanolone only were detected
while in one patient rvho failed to benefit, small but significant amounts of aetio-
cholanolone and androsterone rvere present (Table X). . • j -

Implantation of the pituitarj'^ rvith Yttrium-90 n^as less effective in reducing
the excretion of ll-deoxy 1 7-ketosteroids although in five of twelve patients vhose
cancer regressed and in four of ten patients who had no response, no measura e
amounts of these fractions were present in the urine. In the other patents m
both groups variable amounts of one or more fractions were detected (lable Aip

URIXAKY 17 -KETOSTEROmS IK EKEAST CAKOEH

Fig. 5. — ^Urinary 17-ketosteroid fractions in twenty-tiro patients witli advanced breast cancer
treated by implantation of the pituitary with Yttriuin-90.

DISCUSSION

The estimation of total neutral 17-ketosteroids in female urine is a crude and
relatively non-specific index of adrenocortical activity. In patients Avith breast
cancer their excretion was consistently reduced by adrenalectomy and oophorec-
tomy, irrespective of the cbnical response of the patient, a finding previously

466

SIM, HOBKIRK, STEWART, BLAIR AND FORREST

Table Yl.—Pre-operatwe Estimations of Urinarij n-keiosieroid Fractions in
Patients ivith Breast Cancer Treated by Endocrine Ablation.

17 -ketosteroid fraction

(mg. /day)

Belinviour

Case

1 1 -deoxv

ll-OXJ’

of cancer

number

Endocrine Surgerj-

17KS ‘

17 KS

Ratio : 1

Cancer regressed

Ox 22

Oophorectomy

0-26

(12 patients)

S 20

Pituitarj' implant

0-34

0-49

0-7

Ys 10

Oophorectomy

-pPituitar}' implant

1-20

1-33

0-9

Ys 10

Pituitary implant

1-39

1-15

1-2

S 39

»# It

0-27

0-19

1-4

Ys 13

»* »»

3-10

2-06

1-5

Ys 14

2-33

1-45

1-e

S 44

♦ » ♦»

2-47

0-97

2-5

As fi

Adrenalectomj-
-f Oophorectomy

2-03

1-03

2-6

Ys 27

Pituitary implant

1-24

0-48

3-e

Ox 1

Oophorectom 3 '

5-48

1-62

3-4

As 38

Adrenalectom}-'

-f Pituitary implant

1-55

0-27

5-7

Cancer not improved

Ys 25

Pituitary implant

. 0-26

1-59

0-2 I

(10 patients)

S 1

Oopliorectomj'

Pituitarj' implant

. 2-3G

2-56

0-9

Ys 9

Pituitarj' implant

1-38

1-00

1-4

As 41

Oopliorectomj’

+ Adrenalectomj'

+ Pituitarj’ implant

. 0-44

0-32

1-4

Ys 12

Pituitarj’ implant

1-43

0-94

1-0 ■

Ys 11

Oopliorectomj’

-f- Pituitaiy’ implant

. 3-39

I’Se

2-2

Ys 18

Pituitarj’ implant

. 3-07

1-29

2-4

As 5

Adrenalectomj’

-f Pituitarj’ implant

2-85

0-98

2-9

Ys 15

Pituitarj’ implant

4-29

1-22

3-5

Ys 10

Adrenalectomy

+ Pituitarj’ implant

. 3-35

0-82

4-1

Table VII.-

-Mean Excretion of Urinary 17

■ketosteroid Fractions After

Jlean

ratio

S2-2:l

2 - 1:1

Response to
endocrine
surgerj-

Cancer regressed (10
patients)

Cancer not improved
(6 patients)

11-oxy 17-ketosteroids

Il-deoxy 17-ketosteroids
—

llOHE

0-58

0-49

11 KE
0-92

11 KA
0-02

Total

1-52

0-01

Total

0-01

0-46

0-95

0-04

0-OS

Table VIII.— Jfeaw Urinary Excretion of \1 -ketosteroid Fractions After
Pituitary Implantation ivith Yttrium-90 in 22 Women with Breast Cancer

ll-oxv 17-ketosteroids 11-deoxy I7-ketostero.ds

Response to r-

endocrine 1 1 OH E

surgerj- 11 OH A

Cancer regressed 0-73

(12 patients)

Cancer not improved 0-53
(10 patients)

0-59

0-39

11 KA
0-06

Total

1-38

0-18

0-05

0-06

Total

0-29

0-04

0-96 .

0-23

0-10

0-02

0-35

XmiNARY 17-KETOSTEUOlDS IN BREAST CANCER

•ffi-

Table IX. — Compariso)i of Mean Posloperalivc Excretion of W-oxij \1 -I'closleioifls
in 10 Patie 7 tts Who Besponded and 6 Patient.^ Who Did Not Benpond to
Adrenalectomy and in 12 Patients Who Besponded and 10 Patients Mho did
' Not Bespond to Pituitary Implantation

Nature of
endocrine surgery
Oophorectomy and Adrenal
eetomy

Pituitary implantation

Mean Il-oxv-

Niimlier

. I7-ketosleroid exerelion

Response to

- »
hog.

endocrine surgery

j)nticnts

Arith.

Cancer regressed

i-r,l

i-onn

Cancer not improved .

0-93

0-770

Jlean difference

—

. 0-.78in-

-.40

0-293 -to- 212

—

>0-3

>0- 1

Cancer regressed

I -38

0-9Ifi

Cancer not improved .

0-90

0-839

Mean difference

—

. 0-4194-0-

.709

0-077d;0-J91

—

>0-4

>0-f.

Table X. — The Excretion of Urinary W-deoxy \~-kctosteroids After Adrenalectomy
in 1 IVomen IVith Breast Cancer, hi the Other 9 Patients Studied After
Adrenalectomy and Oophorectomy No W-deozy \1 -ketosteroids IV ere Present

Response to
endocrine

1 1 -deoxy

17 -ketosteroids

surgerj-

Patient

Cancer regressed .

As 17

0-03

As 9

0-01

As 7

0-04

As 38

0-01

As 20

0-01

Ax 4

0-02

Cancer not improved

As 5

0-10

0-1.5

Table XI. The Excretion of Individual U-deoxy 11 -ketosteroids After
Pituitary Implantation IVith in 13 IVomen With Breast Cancer

Response to

endocrine

surgerj-

Patient

Caneer regressed .

Cancer not improved

Il-deoxy I7.ketosteroid.s
— ^

0-20

0-83

0-26

0-45

0-07

0-09

0-04

0- IO

1- 72
0-28
0-02
0-12
0

0-19

0-3,';

0-02

O-II

0-68

0-04

0-19

d
0-10
1-20
0

0-14

0-27

0-01

O-OI

0-17

0-01

468

SIM, HOBICIEK, STEWAET, BLAIE .AKD FOREEST

Table Xll.—Cowipariso?! of Mean Excretion of U-deoxy
Patients Who Res 2 )onded and 10 Patients Who did Not
Ini 2 )lantation

ll-ketosteroids in 12
Respond to Pihiiianj

Response to
endocrine
surgery

Cancer regressed
Cancer not improved

Number Mean Il-deoxy-17-ketosteroids

of ^

patients Arith. Log.

52 . 0-27 0-292

10 . 0-35 0-293

Moan difference
P .

. 0-08I±0-250 0-003iO-lS4
>0-8 >0-9

reported by Strong et al. (1956). In the adrenalectomized patient tlte small
amounts of total 17-ketosteroids excreted in the urine are metabolites of mainten-
ance cortisone, and if this is temporarily stopped they fall to undetectable levels
(Hobkirk ef al., 1959).

In women with breast cancer tvhose adrenals have not been removed ACTH
administration leads to increased excretion of urinary total 17-ketosteroids. This
effect was not noted in any of the adrenalectomized patients studied, indicating
that these patients were without functionally active adrenocortical tissue even
when their cancer failed to respond, a finding which has recently been confirmed by
more sensitive liornione assays (Sim et al., unpublished).

Unlike adrenalectomy and oophorectomy, implantation of the pituitary with
Yttrium-90 did not reduce total 17-ketosteroid excretion either in responding or
non-responding patients. Atropliy of the adrenals after pituitary implantation is
a gradual process and persistent adrenal steroidgenesis may account for the higher
ketosteroid excretion compared %vith adrenalectomized patients (Forrest, Sim
and Stewart, 1960),

Fractionation of the urinary neutral 17-ketosteroids provides a more sensitive
and reliable estimate of adrenocortical function. Our results indicate that the
pre-operative levels of these fractions are not of value in forecasting the response
to endocrine surgery, a finding in agreement with that of Plantin et al., (1958).
Moreover, the5'' do not confirm the recent suggestion by Allen, Hajnvard and
Merivale (1957, 1958), that the ratio of 11-deoxy to 11-oxy 17-ketosteroids in the
urine of patients with breast cancer is useful in this respect, although it must be
stressed that these workers used methods of estimation different from ours and
those of the Stockholm group.

After adrenalectomy and oophorectomy 11-oxy 17-ketosteroids are still present
in the urine provided maintenance cortisone is taken, and they are of value in
indicating persistent adrenocortical function only if maintenance cortisone is with-
dranm (Hobkirk et al, 1959 ; Sim etal., unpublished). After pituitary implantation
also, cortisone is required to maintain good health and metabolites will be excreted
as 11-oxygenated steroids. It is therefore not smprising that the levels of H-
oxy 17-ketosteroids found in the urine after adrenalectomy and oophorectom}"
and after destruction of the pituitary bear no relationship to the clinical response.

The 11-deoxy 17-ketosteroids are mainty metabolites of the C19 androgenic
steroids and their excretion is unlikely to be influenced by cortisone tlierap}
(HolUday, Kellie and Wade, 1960). Their presence in the urine of women wt- i
breast cancer is thus highly suggestive of the preserice of functioning adrenocortica ,
or in the case of aetiocholanolone, possibly ovarian tissue (Plantin et al., I o )■

URINAKY n-KKTOSTEUOinS IX RUEAST CAXCICH

These steroids were completely absent from tlic urine of nine of sixteen iin len s
after adrenalectomy and oophorectomy. In six of tlie rcmaiiimg seven patients
sLll amounts of aetiocholanolonc (0-01-0-04 mg./2-l hr.) were (letcc eil a^
in only one patient whose cancer did not improve following adrciia cctomy, neie
both aetiocholanolonc (0-16 nig./24 hr.) and androst crone (O-lo ing./:.-l hr.) found.
This latter patient did not have any clinical response to siihserpicnt ])itnitnr\
implantation ndth Yttrium-90 (100 per cent destruction) and no adrenal rests

were found post mortem. . , , i • it

Other workers have also described small amounts of aetiocholanolonc m t lie
urine of adrenalectomized-oophorectomizcd patients (Kellie, 19r)4 ; Kellie and
Wade. 1957 ; Bulbrook, Greenwood and Thomas, 1958). With the met hod we
used the quantitative significance of amounts of less than 100 //g. is doubtful
although they can be quantitativelj’^ detected down to a level approximating
10 //g. : it is quite clear that the pre.sence of such small amounts of this steroid
in the urine of adrenalectoinized patients does not preclude benefit from the
operation.

Implantation of the pituitary with Yttrium-90 was less effective in reducing the
excretion of these fractions and aetiocholanolonc (0'02-l‘72 mg./24 hr.) was found
in the urine of twelve and androsterone (0-02-0-68 mg./24 hr.) and ji fraction
(0-01-1-20 rag./24 hr) in the urine of seven of the twenty-two patients studied.
The amounts of these steroids in the urine were not related to the clinical response.

Aetiocholanolonc and androsterone have also been recovered from the urine of
women with breast cancer after surgical hypophj'sectomy (Holliday, Kellie and
Wade, 1958) although these workers could not isolate deh 5 'droepiandrosterone from
the urine of any of the twelve patients studied.

In our experience ll-deoxj'^ 17-ketosteroids ma}’^ still be recovered from the
urine when the intrasellar pituitary is completely destroyed, although the levels
are generally low provided 90 to 100 per cent of the gland is necrosed (Forrest
et al., 1960). It is known that aldosterone secretion persists despite comjilcte
hypophysectomy (McLean et al, 1957) and evidence has also been presented that
cortisol synthesis also may continue in the absence of a functioning pituitarv
gland (Forrest et al., 1960). It does not seem unreasonable to suppose that the
autonomous adrenal may also be able to synthesize Cl9 steroids, albeit in reduced
amount, and further evidence of this has recently been reported bv M^ilson
Lipsett and Butler, (1960). ‘ ’

The presence of 11-deoxy 17-ketosteroids in the urine of women whose breast
cancer had recessed following pituitary implantation clearly indicates that pitui-
tary destruction can produce a beneficial response in the absence of full adrenal
suppression. This would suggest that deprivation of a pituitary^ hormone other
than corticotrophin was primarily responsible for its effect on t^our growth.

breads Y' Y" "'iW'

of tli8 pituitary gland irith Yttrium-90. No ikHomWn ha°aT “f ’Y?"'”*'™
pre-operative levels and the resnonse +n JrirT • ” ^ ^ between

omy and oophorectomy 17-ketosteroids with following adrenalec-

derived from maintenance cortisone persist in the wliicli are

one, persist in the urine whereas those without

470

SIM, HOBKIEK, STEWART, BLAIR AND FORREST

this function are generally absent. Trace amounts of aetiocholanolone may
still be found in the urine of some patients after adrenalectomy and oophorectomy
but this is not of significance in relation to their response to the operation.

Following pituitary implantation with Yttrium-90 both 11-oxy and 11-deoxy
17-ketosteroids are more consistently found in the urine indicating that full
adrenal suppression is not required for benefit from this operation.

We wish to thank Professor C. F. W. Illingworth in whose department this
study iras carried out, for his interest and guidance. This work was supported
by the British Empire Cancer Campaign from whom Dr. Hobkirk, Jlr. Sim, j\Ir.
Blair and Dr. Stewart were in receipt of full-time grants. We also wish to thank
Mr. B. A. McAllister, Miss Eleanor Lunney and Mr. Neal Carlin for technical
assistance.

REFERENCES

Allkn, B. J., Haywasd, J. L. and Mekwale, W. H. H. — (1957) Lancet, i, 49G.
(1958) ‘ Endocrine Aspects of Breast (lancer Edited by A. R.. Currie.
Edinburgh (Livingstone), p. 253.

Bulbrook, R. D., Greenwood, F. C. and Thomas, B. S. — (1958) Biochem. J., 69, 196.
Forrest, A. P. M., Blair, D. W., Peebles, Brown, D. A., Stewart, Helen J.,
Sandison, a. T., Harrington, R. W., Valentine, J. M., and Carter, P. T.—
(1959) Brit. J. Surg., 47, 61.

Idem, Sim, A. W. and Stewart, Helen J. — (1960) Proc. R. Soc. Med., 53, 83.
Hobkirk, R. — (1958) J. cUn. Endocrin., 18, 636.

Idem, Sim, A. W., McAllister, R. A., O’Donnell, V. J., Morris, Sasha, Peebles
Bromti, D. A., Blair, D, W. and Forrest, A. P. 51. — (1959) Scot. med. J., 4,

539.

Holliday, 51. E., Kellie, A. E., Wade, A. P. — (1958) ‘ Endocrine Aspects of Breast
Cancer ’. Edited by A. R. Currie. Edinburgh (Livingstone), p. 224.— (I960)
Acta Un. int. Cancr., 16, 185.

IvELLiE, A. E.— (1954) Rep. Brit. Emp. Cancer Campgn., 32, 464.

Idem AND Wade, A. P.— (1957) Biochem. J., 66, 196.

McLean, J. P., Lipsett, M. B., Li, 51. C., West, C. D. and Pearson, O.H.— (1957)
J. din. Endocrin., 17, 346.

5IEDICAL Research Council. — {1951) Lancet, ii, 2^.

Plantin, L-0., Birke, G., Diczfalusy, E., Franksson, C., Hellstrom, J., Hultbert
S. AND Westman, a. — ( 1958) ‘ Endocrine Aspects of Breast Cancer . Laitea
bv A. R. Currie. Edinburgh (Livingstone), p. 244. t

Strong,' J. A., Bkoavn, J. B., Bruce, J., Douglas, 5L, Klofper, A. and Lobaine,

J. A. — (1956) Lancet, ii, 955.

{1954) Arch. Biochem. Biophys., 52, 217.

Idem, Lipsett, M. B. and Butler, L. C. — (1960) J. dm. Endocrin., 20, 534.

471

PLASMA /9-GLUCIIRONIDASE LEVELS IN EEEAST CANCEE

B. L. WHITAKER

From the Royal Free Hospital, Ix)ndou, Il’.C'.l
Received for ptiblicnlion July 2!1, 1000

It has been established by Kerr and Levvy (1947) and Levvy, Kerr and
Campbell (1948) that there is a connection between tlie level of tissue //-glncnroni-

dase and processes of growth and repair.

Fishman, Kasdon, Bonner, Fishman and Hombnrger (lOf)!) have slmwn that
alterations in the hormonal state of the subject may have a profound effect on
the level of the enzyme in the blood and tissues.

The blood level of ^-glucuronidase in 23 patients with breast cancer has been
investigated by Cohen and Huseby (1951 ) who found the difference between normal
controls and cancerous subjects to be barely significant.

Goldbarg, Pineda, Banks and Rutenberg (1959) however found raised blood
levels in 14 out of 21 cases of breast cancer especially in those with liver second-
aries.

Boyland, Gasson and Williams (1957) observed high /J-glucuronidaso levels
in the urine of patients with malignant disease, including carcinoma of the breast.

The present work deals with the plasma ^-glucuronidase levels in a scries of
47 patients suffering from malignant disease of the breast and also with factors
which may have influenced these levels.

METHOD

Blood was taken into oxalate bottles, the plasma separated as soon as possible
and stored in rubber stoppered glass tubes at —20° C. for periods of 1-5 days.
In order to minimize diurnal variation the specimens were collected whenever
possible at the same time of day, namely 1.30 p.m.

The loss of activity of the enzyme due to storage was of the order of 10 per
cent over 7 days. ^

The method of estimation has been that of Tallalay, Fishman and Hugmns
(1946) as modified by Boyknd, Wallace and Williams (1955) and Boyland, Gasson
and Wilhams (195/ . The substrate, phenolphthalein mono-/?-gIucuronic acid
(0-05 g. Sigma) was dissolved in 20 ml. ethanol and diluted to 100 ml. with water
The plasma specimens were diluted 1 in 10 with water immediately before incuba-

per hour at 37° C. ^ P^^i^olphthalein per ml. of plasma

472

B. L. WHITAKER

MATERIAL

Fifty normal women and 47 patients suffering from breast carcinoma were
studied. A total of 368 estimations was performed on these subjects both before
and after treatment. One hundred and ninety-three of these estimations were
performed before the patients received treatment (apart from androgens ivliich in
many oases had been given before the patient was referred, and operative pro-
cedures such as removal of the primary lesion which in some cases had been
performed many years before) and these 193 estimations form the basis of this
report. It is lioped to publish the findings with regard to the effect of treatment
on the enzyme levels in a later report.

1. Normal controls . — Fifty normal women aged from 18 to 70 years served as
controls (Table I). Thirty -four of these were hospital patients about to undergo
operation for hernias, varicose veins, etc. The remainder were students and
members of the hospital staff. Thirty of the 50 were premenopausal and 20
menopausal or postmenopausal. The overall mean value of ^-glucuronidase for
these 50 individuals was 3-58 units and the standard deviation 1-31. The normal
range was taken as from the mean, i.e. 0-96 to d-20 units.

The mean values for the pre- and postmenopausal groups were almost identical
being 3-57 and 3- 69 respectively.

2. Cases of breast cancer . — The enzyme levels and relevant clinical details for the
47 cases of breast cancer are summarized in Table 11. The mean ^-glucuronidase
for this group irrespective of staging or previous therapy is 6-90 units (standard
deviation 3-87). This figure is significantly greater than the mean value of the
control group (c = 5-63 P = a 0-0000001). Fifty-one per cent of these cases
had enzyme levels within the normal range and 49 per cent had elevated readings.

Analysis of cases

An anaij^sis of these figures was undertaken in an attempt to correlate them
rvith the clinical and pathological findings in individual cases. The results are
expressed graphically in Fig. 1.

(a) Clinical staging . — Of the 47 cases 10 (21-25 per cent) were clinically classed
as stage I or II (Table II). The mean value for this group is 6-13 units (standard
deviation 4-07). A comparison of this with the control group shows the difference
to be significant [t = 3-67 P = < 0-001).

The remaining 37 cases were classed as stage III or IV and the mean value
for this group is 7-11 (standard deviation 3-84). Comparison with the control
group gives a value for c of 5-35 and for P of < 0-0000001.

Comparison of the stage III and IV group with the stage I and 11 poup
however shows a difference in means of only 0-98. This does not attain statistica

significance (i — 0-71 P = < 0-5).

(b) Previous hormone therapy . — Of the 37 cases in the stage III and 1\ gro p
20 had received treatment with androgenic hormones usually methyl testosterone
by mouth or testosterone propionate by injection at some time in the course o

their disease. , 07

Cases 31, 33, 34, and 38 in the androgen treated group and 32, 35,
in the non-treated group had clinical evidence of liver involvement at the time
of the estimations and are excluded. This leaves for comparison rea
13 untreated cases. The mean values of /?-glucuronidase for these two gro p

GLUCLTRONIDASK LEVKLS IK inuiAST {;aX(MCK .,7;,

does not indicate a^i^nSn^^iff^^^^^^^ availal.le (l.is

groip haT.;: '■" ' ' '•

either enlarged livers iaimrlino r./ i i i- damage, ^^in(> cases had

(No.2c)obsS™!;Z;4Jr,,Zcte^^ 'r;

liver metaataais a„<l aha ia tj:crcr„,-c incl,„le,rin M

Tablk I. — Normal donlroU

Control

number

4
o
6

8
9

20
21
22

45
40

Age

30
37
22
IS
21
22
28
25
32
32
27
22

31
62
63
44
55

52
55

53
63
65

40
70
21
55
21
21
21
18
62
52
28

54
57
29

39
28
22

40
57
51

39
61
29

llcintion to
inonopiiiiso
I’roincnojnitisnl

Postinenopnusnl

Premenopnu.*!nl

Postmcnopau.sn!

Menopausal

Postmenopausal

Menopausal

P ostmenopausal

Premenopausal

Postmenopausal

Premenopausal

Menopausal

Premenopausal

P ostmenopausal
Menopausal
Premenopausal
Postmenopausal

Premi

ienopausal

Postmenopausal

Premi

Postm

enopausal

Prem:

enopausal

ostmenopausal
premenopausal
i'ostmenopaasal

Kum

remi

)er of
iiigs

.lleiin

/t-gliicuronidau
4 • 30
1 • 85
1-27

1 - 40
3-13

2 - 25

2 - 48

3 - 24
3-40
3-16
3-51

t> ^ m

r,}

5-01

3 - 86

4 - 53
2- 75
2-32

1 - 97
4-87
4-60

2 - 35

3 - 80

3 - 30

1 - 67

4 - 05

5 - 00

2 - 44

3 - 95
3-66
3-86
3-90

•32

•60

'20

8-50

4-85

4 - 20
2-44

5 - 05
4-10

2 - 73
4-00
3 • 86

3 - 30
2-00

4 - 01

474

B. L. WHITAKER

The mem /J-glucuronite level of the group without liver seconderies w.s
6-93 Md of the other group 7-26. TJ* difference ie not sigmBoant (( = 0 34

nno hepatic group were actually jaundiced at

one stage of their disease. The mean value for this group (No. 26, 32, 34, 37 and

Table II . — Breast Cancer

Case

number Ace
1 . 80

2 . 48

3 . 55

4 . 71

5 . 68

6 . 59

7 . 71

Relation

Androgen Hepatic

Humber

read-

Mean

/5-glucu-

menopause

therapy metastasis

Histology

ings

ronidase

Post.

Spheroidal

IO-47'I

Men.

. 1 .

14-70

Post.

4-85

Poorly diff.

5-91

*f >»

3-70

Mucoid.

2-42

»*

Scirrhous

1-85

. 48

Pre.

—

Spheroidal

. 34

—

. 34

»>

—

Anaplastic

. 47

Hot known

. 49

—

Undiff.

. 55

Post.

Spheroidal

. 70

»» *

•4"

—

. 51

»♦

-f

—

Scirrhous

. 54

Pre.

—

Hot knon-n

. 35

—

Undiff.

. 52

Men.

—

Scirrhous

. 42

Pre.

—

Anaplastic

. 46

Rnd. men. .

—

. 79

Post.

. 37

Rnd. men. .

-f

—

. 78

Post.

—

Hot known

. 58

—

Scirrhous

. 73

ff ♦

—

. 32

Pre.

. 56

Men.

■4~

Spheroidal

. 65

Post.

—

Not kno™

. 49

Pre.

—

Spheroidal

Post.

—

Anaplastic

. 47

Pre.

Not knon-n

. 48

Post.

»» »'

. 54 .

Scirrhous

. 52

Not known

. 55 .

Scirrhous

58 .

—

Paget’s and
scir.

53 .

Not known

43 .

Post-hjqjo. .

Anaplastic

47 .

Pre.

—

Scirrhous

65 .

Post.

—

Not known

58 .

—

Spheroidal

scirrhous

52 .

4”

Mucoid

59 .

»»

—

Not knoivn

45 .

—

»♦ »»

63 .

—

»> y>

62 .

—

>» >' •

54 .

—

Scirrhous

simplex

266 .

4“

7-70'|

2-43 >Stag 0 II

2- 55'
1-90

3- 50
5 -87

4- 84
18-62
18-80

5- 05
9-00
7-32
4-12

11-50

7-52

5-27

5-79

4-32

3-67

10-25

11-25
4-05 r
6-58
4-73

Stages III
and IV

7-99

6-60

4-72

8-83

12-90

9-85

4-36

4-57

4-33

8-80

9-44

7-00

6-66

4-00

6-35

6-62,

GLUCUROXIBASE LEVEES IX UUEAST CAXCEH

38) dimug the jaundiced period was S-r,h which of cf)urso. for so few cases is not.

niitside the ranee of chance deviation. _

(d\ Hisloloql —T \\g histology of the lesion was known in .LI of the •( < c ...
Twin V one of tliese were described as anaplastic and the retnninder were sen-

.logrccH of Tl.; me.. v„„.o» ..f

glucuronidase for the two groups arc /•41 and irlG (t - l-d.. 7 L

Fig. 1. — Comparison of plasma ^-glucuronidnso levels of 50 normal women with 47 en.^tes of
breast cancer. Tlie dotted horizontal line represents the mean value for the control group
and the upper and lower continuous lines two standard deviations around this moan.

DISCUSSlOIv

The difference in levels between cancerous and non-cancerous individuals in
^is series is highly significant. This corresponds with the findings of Goldbarg,

Pineda, Banks and Rutenburg (1959), but not with those of Cohen and Huseliv
(1951). •’

The reason for this discrepancy between different workers is not clear. The
method used by Cohen and Huseby (1951) is that of Tallalay, Fishman and
auggins (1946) while Goldbarg, Pineda, Banks and Rutenburg (1959) iised 6-
^°™°’^'^^phthyl ;?-D-glucopyTuronoside as a substrate.

TalkbTv™?^'^ ^ modification of the method of

suggested by Boyland, Wallace and

The estimations have been carried out on oxalated nlasma withniTf o i iv
precipitating agents such as the

476

B. L. AVHITAKER

SnttlTosS)'’ ““ “““ Pi.>eda. Eanfe .„„

The difference in means between the stage I and II group and the staop ITT
outiS sufficiently large in relation to the number of cases “to ^be

outside the range of chance but serial observations on individual patients have

n ^ corresponding to deterioration in clinical

state. Such a case is illustrated in Fig. 2.

with advance in disease process.

It seems likely that with larger numbers of cases this gradient ivill attain signi-
ficant proportions.

The findings with regard to hepatic metastasis do not correspond with those of
Goldbarg, Pineda, Banks and Rutenburg (1959) who found raised levels in a
majority of cases of hepatic metastasis from breast cancer. Though in the
5 jaundiced cases very high levels were seen which suggest again that vlth a
larger number of cases the difference might be significant.

The correlation with histological grading is of interest, suggesting as it does,
the possibility that there may be less ^-glucuronidase activity in those patien s

with scirrhous types of groudh. . ,

The question of hormonal effects on tumours and on y9-glucuronidase is o

477

GLUCUBOXIDASE LEVELS IX HUEAST CAXCEH

considerable complexity and these results do not ns yet allow of any hut llie
most tentative suggestions as to their relationship. It is iierliaps of inleiesl.
however, to note that the ratio of normal to abnormal levels m the cancer group
{51 and 40 per cent) is not very far removed from tlic pereentages given by Ihiron.
Curling and Eadley-Smith (1058) and Luft, Olivccroim, Ikkos, ^II.«son and
Ljunagren (1056) for remissions and failures following h}i)Oi)Iiysectouiy. I Ins
may “of course be entirely coincidental hut investigations are in jirogress to
ascertain whether any relationship exists.

SU.MMARY

(1) An investigation into the plasma /?-ghicuronidase levels of 47 cases of
breast cancer and 50 normal women has been undertaken using a modilication of
the method of Tallalay, Fishman and Huggins (1046).

(2) The difference between the enzyme levels of the cancerous and non-
cancerous groups was highh' significant.

(3) Stage I and 11 carcinomas were associated with significantly raised enzyme
levels. Stage III and IV cases had higher mean values than Stage I aiul 11
but the difference was within the range of chance.

(4) Cases who had received androgen therapy had somewhat higher mean
values than those who had not had androgens and those with anajilastic growths
had higher values than those with scirrhous but chance variation cannot be ex-
cluded as a cause for tliis.

(5) No great difference in enzj’me levels could be demonstrated between cases
with and without hepatic metastasis in the absence of jaundice.

I nish to e.xpress my thanks to Mr. E. J. Radley-Smith and Dr. D. N. Raron
for sponsoring this work and for their advice and encouragement : also to Dr.
D. C. Williams whose very considerable experience in this field has been of the
greatest help in overcoming technical and other difficulties ; to the Consultant
Staff of the Royal Free Hospital who have generously allowed me access to their
patients ; and to Sister Hitchcock m’thout whose help and co-operation the
collections of specimens would have been impossible.

Financial support for the apparatus and material used in tlie investigation was
generouslj’" provided by the British Empire Cancer Campaign.

REFERENCES

Babox, D.N., Guelixg, K. J. axuRadley-Sjuth, E. J.— ( 1958)Rn7 .7 Sum 45
Boa-uaxu, E., Gassok, J. E. axd WmuiAAis, D. G.-(19.57) BhI j

Idem, Vv^UACE, D. M. Axn WiLurAALS, D. C.— (195.5) /ftjf/., 9 62. ' ’

’ w HnsEBY, R. A.— (1951) Proc. Soc. exp. Biol. K.Y. 76 304
Fishm^-, 11 . H., Kasdox, S. C., Boxxek, G. D., Fishiiax L W avti

F.-(1951) .7. din. EndoerL, 11, 1425. Ho.mburgek,

GoMTO(ero(o^36, isf' ® EcTESBrao, A. M,_(ln59)

Lmi, E., Om-EcBOKi, i,, 42, 462.

*4?Her. J. Med., 21, 728. >u.ssox, L. axd Ljuxggkex, H.— (19,50)

T.vleaeay, F. EisHYiv W vr TT ^

, Xisn-MAX, W . R. AXD HugGEXS C— HOAfil T k- i
34 -J- biol. Chem., 166, 757.

478

THE HISTOGENESIS OF aiALIGNANT TTOIOUES
INDUCED BY COBALT IN THE EAT

J. C. HEATH

From tU Strangeways Sesearch Laboratory, Cambridge
deceived for publication May 31, 1960

The production of malignant tumours in rats injected intramuscularly nitli
pure cobalt metal powder has been described already (Heath, 1936). Tumoius
occurred at the injection site in 17 out of 30 rats over a period ranging from 5 to
12 months after the injection. Tliirteen out of the 17 tumours contained a
malignant component derived from muscle ; malignant connective tissue ele-
ments were also present in some of these 13, and in the remaining 4 tumours
the malignant process appeared to have arisen predominantly in the connective
tissue. The tumours were rhabdom 3 '’osarcomata, rhabdomj’'ofibrosarcomata,
pleomorphic and fibrosarcomata.

The purpose of the present investigation was to follow step step and in
some detail the tissue changes caused b}’’ the injection of powdered cobalt metal
into rat skeletal muscle, up to the stage at which ffanldy malignant changes could
be recognised.

SIATEEIALS AND METHODS

Two experiments were made. In the first the rats were killed at fortnightly
intervals ; since the results showed that pronounced changes were ahead}’’ present
in the tissue at 2 weeks, a second experiment was undertaken in which the animals
rvere killed at intervals of 1 to 28 days after injection.

Male rats of the hooded strain aged 2-3 months were used. Tliirty animals
were injected in the thigh muscle of the right leg with 0-028 g. cobalt metal powder
(spectrographically pure) shaken into suspension with 0-4 ml. of fowl serum :
15 controls Avere similarly injected with 0-4 ml. fowl serum only. At intervals
after the injection animals taken at random were killed by cervical dislocation,
and the portion of the thigh muscle surrounding the injection site was excised.
Pieces of the excised muscle were fixed in Carnoy’s fluid for subsequent staining
with methyl green-pyronin (MGP), and in Zenker’s fluid for Azan staining .
tissue fixed in Carnoy’s fluid, however, also gave satisfactory results with Azan.

BESDETS

As in the previous experiments, little or no local reaction to the injection could
be detected by examination of the intact animal, and there were no genera ox'C
effects. Study of the histological material, however, revealed a distinct ana
characteristic chain of events in the cobalt-treated rats but nothing of consequence
in the controls injected with serum only. The progressive changes in tie ^
treated animals led from the initial local trauma, tlirough a stage of cell pro i e ■
tion up to the malignant change and the final development of tumours o m yp
previously described (Heath, 1956).

MALIGKjVKT tumouks induced dy cobalt

4"ft

Tliprp antiear to be two main processes at work : , , i 4 1

m The ^response of the muscle to the mechanical injurj^ jirocluced by t he
infection of the metal grains, as shown by an attempt at ^‘'‘Sencralmn and rejm
^ (2) The modification of the regenerative and repair process by
actton of the cobalt presumably either by slow solution m tlie tissue {luids to go c
cobalt ions, or by direct catalysis at the surfaee of the metal grams

These two processes are clearly seen in the histological material and arc .>
similar in aU rats affected. In a few animals the injurj^ heals normally, and in
others which are not considered in detail here, the progressive changes leading
to malignancy occur in the connective tissue alone.

The first response of the muscle to the injection appears at 1 day as an in-
filtration of leucocytes into the spaces between the muscle bundles and fibres, m
regions near the primary injurj’^ (I^ig- !)• This infiltration is still seen at 4 days
but by this time many fibroblasts have appeared in tiie region. Large aggrcgntc.s
of cobalt grains can be found in immediate contact with intact muscle uliich
sometimes shows no evidence of damage (Eig. 2), whereas at sites distant from the
cobalt some muscle fibres contain a greatly increased po])ulation of nuclei (Fig. :i).
A nucleosis of this type has been described hj' Albschnl (15)47) who attributes it to
the loss of equilibrium in pressure between the nuclei and the .sarcopla.sin and
fibrillae consequent upon the iniur3\ In other damaged muscle fibres both nuclei
and striations have disappeared to leave a homogeneous liyalinc material.

The muscle continues to degenerate and at 7 days necrotic unstriated bundles
often lie between bundles of normal striated fibres (Fig. 4) ; a further brcakdonoi
of this hyaline substance into amorphous granular residues now begins. Tlie
zones between adjacent muscle fibres, particularlj' where one filire is necrotic,
become filled rrith cells of many tjqies, leucocytes, fibroblasts and characteristic
fusiform cells. These last (Fig. 5) are sometimes mononucleate and sometimes
have 2-5 nuclei ; their cytoplasm is veiy^ basophilic and stains deeply with
pyronin. They appear to be myoblasts, possibly derived from the injured
muscle fibres by the process of “ dissociative degeneration ” descrilied by })rcvious
authors (Pfuhl 1937, Betz 1951, quoted by Godman 1957); this process is .said to
involve the freeing of nuclei from a muscle fibre, each with its own complement of
endoplasm, to form mononucleate myoblasts. In other areas there are multi-
nucleate cell tubes (Fig. G) the cytoplasm of which scarcely stains with prTonin
which correspond to the ceU tubes of Waldeyer (Waldeyer 1865, quoted by Godman
)95i) and represent collapsed muscle fibres. Regeneration has tlius begun.

Extensive areas of degeneration are still present at 12 days ndth increasing
amounts of the amorphous granular material, but regeneration is now well under
way ; in some zones many long multinucleate basopliilic or striated muscle
straps are seen (Fig 7) rvhich sometimes at least are continuous with dama^^ed

4S0

J. C. HEATH

Godman. Fusiform myoblasts (Fig. 8) continue to be formed but tier show
progressively less tendency to associate and mature into differentiated fibres
V\ liereas in the muscle infarction undifferentiated myoblastic elements diminish in
number after 16 days, in the cobalt-treated material they continue to increase
At 4 weeks changes due to the action of the cobalt have spread further from
the metal deposit to more distant regions of the muscle (Fig. 9-15). The cobalt
granules which are still present are usuall 3 r surrounded by a narrow band of
imcrotic niaterial containing p 3 mnotic nuclei and also some degenerate muscle
(Fig. 9). Two or three cell widths away there is a broad zone of viable cells of
various t 5 'pes but mainly leucocytes and fibroblasts (Fig. 10) be 3 mnd which are
viable muscle fibres (Fig. 11). At this stage the changes in the muscle fibres
are much more readil 3 ’' followed than in the earlier material and the histological

EXPLAHATIOH OF PLATES

Fig. 1. — Infiltration of leucocytes into the space between muscle bundles and fibres. One
day after injection of cobalt. Stained Methyl-Green and PjTonin (MGP). X450.

Fig. 2. — Grains of cobalt metal in intimate contact with apparentlj- undamasfed muscle.
Four days. MGP. x 450.

Fig. 3. — Increased numbers of nuclei at a site distant from the injected cobalt but in the
same muscle. Four days. MGP. x 450.

Fig. 4. — Damaged muscle fibre showing its hyaline, almost homogeneous nature, in contrast
with the neighbouring undamaged fibre. Seven days. MGP. x 450.

Flo, o. — Fusiform cell with very basophilic cytoplasm staining deeply with pjionin and
having possibly 5 nuclei. Seven days. MGP. x 450.

Fig. 6. — Jlultinueleato cell tubes in which the ej-toplasm scarcely stains at all with pjTonin ;

these are the cell tubes of IValdoj’er. Seven days. MGP. x450.

Flo. 7. — Region of regeneration containing numerous multinucleate cells of both tipes,
some m'th cross striations. Twelve days. MGP. x200.

Fig. S. — ^Free myoblast, possibfr binucleate, with Cidoplnsm staining deeply with pjuonm.
Nineteen days. MGP. x456.

Fig. 9. — Cobalt granules still present surrounded by a narrow band of necrotic material
containing pycnotic nuclei and also some granular degenerate muscle substance. Four
weeks. MGP. x950.

FtQ. 10. — Very cellular zone of considerable width lying between cobalt grains and intact
muscle fibres. Four weeks. MGP. x950.

Fig. 11. — ^Abutment of the cellular zone upon the intact muscle fibres. Four weeks. MGP.

X950. . . ,

Fig. 12. — ^Abnormal fibres with strings of peripherally situated nuclei. Four weeks. MGP.

X950. , ,

Fio. 13. — ^Damaged muscle fibre with a collection of peripheral nuclei -wrinklmg up tne
sarcolemma. Four weeks, Azan. X950.

Fig. 14.— Damaged muscle fibre with a chain of nuclei disturbmg the regular pattern oi
striation. The nuclei and nucleoli are larger than normal and stain much more deeply.
Four weeks. Azan. x 950. .... , ,

Fig. 15. — Nuclei in affected muscle showing polar caps of pjTonin staming endoplasm, i our

Fig. 16.— Two myoblasts in a region containing much collagen. Note the densely staining
nucleoli. Six weeks. MGP. X950.

Fig. 17. — Binucleate myoblasts, and a mitosis. Ten weeks. MGP.

Fio. 18. — Giant cell similar to those found in the established cobalt-mdueed tumours.

Fourteen weeks. MGP. x950. „ . -.i/t-D voan

Fro. 19. — Two myoblasts showing cross strintions. Fourteen weeks. MGP.

Fig. 20.— Attempt at production of differentiated muscle fibre m the transplanted coba t

F,^ 2 *LD^nera^to'f'mu’^clf'.™bstance: graatoar pi^ented daposifs atototog from

nodule as shown in Fig. 21. Twenty weeks.

Fig. 23. — ^Mitosis in giant cells in the same
MGP. X950.

British .Iouhkai, of Canxkh.

Voi. xrv. Xo. n.

Hcatli.

▼ »

jrALIGNANT TUMOURS INDUCKU R^ CORAUI

•US I

picture lends support to the view that the mononucleate myohlasts are clemTd
from mature fibres bv the dissociative degeneration mentioned al) 0 \c In (
cobalt-treated material muscle fibres closest to the cobalt implant difTcr from
normal fibres in the presence of strings of nuclei along the iicnphcn (i ig. 1-) .
the nuclei may be so close to the surface of the cytojilasm that the sarcolcmma is
M-rinlded and the striations disturbed giving the appearance of an outpouring ol
nuclei from the interior of the muscle fibre (Fig. Kl and U). In some areas both
the nuclei and nucleoli in these strings are larger and more deeply staining tiian
normal (Fig. 14) ; other nuclei are associated with large jiolar cajis of basopliilic
pyronin-staining material (Fig. 15) the so-called endoplasm referred to nl)o\c.
which according to Altschul (1947) is the true cytoplasm of the muscle cells and
independent of the sarcoplasm and fibrillae of the musclc-fibre iirojicr. This
dedifferentiation ” of mature muscle fibres continues under the action of cobalt
whereas an isolated mechanical trauma only evokes a limited degree of di.ssociation
sufficient to repair the damage (Godnian, 1957). Free inj'oblasts ajipcar in t he
zones near the cobalt injection ; at 6 weeks these cells are larger than in the initial
stages of the tissue response, assume more and more abnormal forms, and usually
have very basophilic cytoplasm and large deeply staining nucleoli. I’hcy may
be present between existing muscle bundles, in the residual necrotic material, and
in masses of collagen (Fig. 16).

At 8 to 10 weeks free myoblasts are verj’- abundant and many are binuclcate.
Mitoses are now seen but whether in myoblasts or other cell types has not been
established (Fig. 17). At 14 weeks giant cells (Fig. IS) appear in increasing num-
bers and although their origin is not certain, similar cells are common in the fully
developed cobalt-induced tumour. Sfyoblasts abound, and while some show
cross-striations (Fig. 19) most do not.

The continuing action of cobalt on the newly formed myoblasts probabl\'
prevents them from redifferentiating into striated elements as* they do in repair
after infarction. That some capacitj’’ for redifferentiation may persist at least
in some cells is suggested bj' the fact that multinucleate straps occasional!}' appear
even in cobalt-induced tumours that have been transplanted for manv Rcnerations
(Fig. 20).

At 16 weeks muscle substance continues to degenerate into pigmented granular
masses (Fig. 22) and mitoses are now verj' frequent, in spite of the fact that cobalt
IS stiU present ; they even occm in the largely necrotic regions adjacent to metal

^ains. In rat cobalt is still present at 20 weeks and a tumour nodule is just
discermble (Fig. 21, 23). •'

seems clear that it is from myoblasts of lh:=; h..!! i i T ' . ^

the breakdown of mature fibres that tRo r > probablj’- originating through
in ton develop

entiated tissue is veiy^^SY\o^fblk,w^^"^]f^'^ breakdown of such a highly differ-
or such tuusou^

482

J. C. HEATH

lias not been often reported. The fact that the carcinogen in these studies is an
element, the metal cobalt, and therefore can be followed throughout the organism
by various chemical and tracer techniques -ivill enable the investigation to be
pursued further in several directions, and gives some grounds for hope that the
nature of one particular carcinogenic mechanism can be found.

The work of several authors has shown that cobalt can inhibit the respiration
of various tissue and cells. Unpublished experiments by my colleagues, Dr. M.
Webb, Mr, J. T. Dingle, Dr. M. R. Daniel and myself, now in progress in this
Laboratory, show that cobalt is a poison for some respiratory systems of rat
tissues. Experiments are being made to find whether there is a correlation be-
tween the dedifferentiation of muscle tissue described here and the inhibition of
respiration, and if so what is the nature of the connection.

SUMMARY

The injection of cobalt metal powder into the thigh muscle of rats regularly
produces a high incidence of characteristic malignant tumours, many of which
are detived from the muscle tissue itself. The steps in this process have been
follou’ed and found to be very similar in different rats. The carcinogenic process
appears to be firstly an extensive and continuing breakdown of the differentiated
muscle fibres into free ra3mblasts, and secondly the transformation of some of
these myoblasts into malignant variants. A possible mechanism is suggested.

The author is deeplj’’ indebted to his technical assistants, Mrs. Audrey Thomp-
son and Miss Angela Orledge for their skill and patient attention to detail without
which this work ^\'ould not have been possible. He also wishes to thank Dr.
Honor B. Fell, F.R.S. and his other colleagues for the many helpful and stimulat-
ing discussions which have played their part in the interpretation of these findings.

The work was financed by’’ the British Empire Cancer Campaign.

REFERENCES

Altschul, R. — (1947) Hev. canad. Biol., 6, 485. , , r< i

Betz, H.— (1951) Arch. Aiiat. niter. Morph, exp., 40, 46 (quoted by Godmanp
Firket, H.— (1957) Thesis. Faculte de Medecine. Universite de Liege. Recherclies
sur la synthese des acides dfeo.xyribonucleiques et la preparation a la nntose
dans des cellules cultivees in vitro ’.

Godman, G. C.— (1957) J. Morph., 100, 27.

Heath, J. G.—{195Q) Brit. J. Cancer, 10, 668. , , ^ , ,

Pfuhl W. (1937) Z. mikr.-anat. Forsch., 41, 569 (quoted by Godman).

Waldeyer. W.— (1865) Virchoivs Arch., 34, 473 (quoted by Godman).

-183

miT OOTOtlR AMD EXPEKMENTAL MELANOTIC TUMOOK
COAT COLOTO HAMSTER

0. ILLi\IAN A>-D F. N. GHADIaVLLY
From the Department of Pathology, The University. Shcfjidd

Keeeived for publication Juno 2, IPGO

It has been shotm that multiple cutaneous melanotic ^^monrs are reachly
-produced in hamsters painted ndth chemical carcinogens (De la Poita cl ah, lO.jG ,
Shubik, etah, 1956; Horning, 1958; Ghadially, 1959) and that these tumours
arise from a net-n'ork of melanocjdes surrounding some of the pilosehaceous follic .
(Ghadially and Barker, 1960). Hitherto only the common brown variety (often
referred to as the golden) of the S3Tian hamster has been einployed for cutaneous
carcinogenesis. Two other colour varieties, cream and tvhite, are also now coin-
mercially available. It was decided to search for melanocjdic netu-orlcs m tlieir
skin and attempt to produce melanotic tumours by repeated applications of
9 ; 10 dimethjd 1 : 2 benzanthracene (DMBA).

JIATERIALS AND METHODS

Normal hamster shin. — The skin of six bromi, six cream and six wliite hamsters
aged 6-12 months was examined. In each colour group there were equal numbers
of males and females.

Preparation of skin for examination. — ^After clipping and close sliaving, the
skin on the flanks and dorsum was removed and pinned out on a piece of cork.
The subcutaneous tissues were dissected off and the skin floated on 4 per cent
formaldehyde for 24-48 hours. The skin was then removed from its cork mount,
dehydrated in alcohol, cleared in xylol and mounted in balsam. In some cases
the skin was returned to water stained rvilh haeraatoxylin and eosin, dehydrated,
cleared and remounted in balsam. In such whole mounts of skin melanocjdic
networks can be easily identified under a low power binocular microscope. This
technique is preferable to routine sectioning since it allows the inspection of a large
area of skin. Some whole skin preparations were subjected, to the dopa reaction,
gold chloride and Fontana’s silver method for melanin. In each instance results
were unsatisfactory as the specimens became too opaque for microscop3^

Histological sections . — ^Pieces of skin and tumour rvere fixed in 4 per cent
formaldehjde and sectioned and stained with haematoxj^lin and eosin in the usual

manner. Some of these sections were also stained with Fontana’s silver method for
melanin.

Production of tumours.— broum, 24 cream and 24 white hamsters
aged 4-6 months and weighing about 90 g. were used in this experiment In

numbers of males and females.^ The hair on
the flanks of all these animals was removed rvith electric dinners A 9 -nor n f
solution „t DMBA in acetone ivaa applied avith a oS liS brael, t„ ffl T
,n„„cd..tely antrennding the left ooeSvertebral spot.2« p«Se^e^

484

O. ILLMAN AND P. N. GHADIALLY

^ a majority of melanotic tumours

SLTf* ”® situation. A constant painting technique was established and
adliered to so as to ensure that each animal received approximately the same
amount of carcinogen. Animals were painted once a week for a period of
weeks and observed for a further period of 9 weeks. The number of melanotic
tumours seen in each animal was recorded at weeldy intervals during the period
of painting and at 2 or 3 weeks interval in the later stages of the experiment.

KESULTS

Normal hamster skin

(a) Costo-veiiebral spot.— The costo-vertebral spot in the brovm and cream
variety is well pigmented and easily recognized. In the white variety it is pink
and difficult to distinguisli from surrounding skin. In whole mounts of skin the
C.V. spot of the bromi and cream hamster shows large heavily melanized melano-
C 3 'tic networks. In the ivhite variety the melanocytic networks are difficult to
distinguish from the surrounding tissues. Their presence is however confirmed
bj’’ the occurrence of a few melanin granules in the expected site, around the
pilosebaceous apparatus in some of tlie specimens. Thus it would appear that
in the white hamster the melanocytic networks are present but are difficult to
detect because thej^ are hypomelanotic or at times virtually amelanotic.

(b) Small pigmented spots . — In addition to the two large pigmented C.V.
spots numerous small pigmented spots have been observed by Ghadially and
Barker (1960) in the sldn of the brown variety of the Syrian hamster, and it was
shorni that nielanocjdic tumours arise from these structures. These small black
spots are produced by a collection of melanocjdes around some of the pilosebaceous
follicles (see Fig. 2 and 3 of Ghadially and Barker, I960). We were able to confirm
tlie presence of a large number of these structures in all the six brown hamsters
we examined, but were able to find onty a few small black spots in the six cream
liamsters. In the six white hamsters no such spots were detected.

3Ielanotic tumours

Many melanotic tumours developed in the brown and white varieties but none
were seen in the cream variety (Fig. 1). Table I shows the rate at which the
tumours appeared in the brown and white varieties. It can be seen that
melanotic tumoims developed earlier and in greater numbers^ in the brorm than
in the -white variety. Further it was observed that tumours in the white variety
increased in size more rapidly ; thus ultimately the white variety showed fewer
but much larger tumours (Fig. 1). It was also noted that both in the broum anc
■white hamster some of the small melanotic tumours regressed after painting hact
ceased, while others were destrojmd by expanding squamous cell carcmomas wliicli
developed in some animals. An analysis of otrr results (Table II) shows that raanj
more melanotic tumours developed in females than in males.

Microscopic examination

Almost all the tumours in the browi variety were abundantly
appeared jet black in colour (Fig. 2) ; only an occasional grey
tumour -was found.

melanized and

hypomelanotic

IvIELAKOTIC TUMOURS IN HAMSTERS

485

Table l.-Rate of Melanotic Tumour Production vi Brown and White Hamsters
Painted ivith 9 : 10 Dimethyl 1 : 2 Demanthacenc

Time

Number of
surviving
animals

Number of
animals

showing tumours

Total number
of tumours

Percentage of
survivors
showing tumo\irs
- ■>

Average numl)er
of tumours
l>or animal

■

weeks

Brown

"vTOtc

Brown

White

Brown

White

Brown

White

Bromi

White

. 23

.33-3

33- 3

34- 8

on -6

G9-0

73-n

73-8

94-7

12-5

20-8

29 "2

41 -7
70-8
79-1

I-O

1- 3

2- 0

1- 5

2- 3

2-4

2-3

1-3

1- 4

2- 1

3-1

2-8

3-2

1 5

94-7

79- 1

3-3

3- 1

,18

94-7

79-1

4-2

3- 1

/ 19

100

78-3

4-5

3- 1

100

78-3

3-6

3-2

100

78-3

4-6

2-8

103

100

78-3

5-3

100

78-3

5-1

3-9

2-3

100

88-2

0-3

2-7

. 12

100

88-2

5-8

2-8

. 12

100

88-2

7-8

3-5

100

94-1

7-8

3-3

Table

Time

weeks

9
10
11
12

20
21
22
24
20
29
31

II . — Bate of Melanotic Tumour Production in Male and Female Hamsters
Painted with 9 ; 10 Dimethyl 1 : 2 Benzanthracene

Number of
surviving
animals

Number of
animals

showing tumours

Males Females Males Females

Percenfago of Average number
Total number survivors of tumours

of tumours showing

Males

Females

Males

12-5

21-7

52-2

56*5

65-2

73-9

90-0

89-5

100

89-5

85 -7

85-7

105

85-7

92-3

tumours

per

animal

Females

Males

Females

20-8

1-0

20-8

1-7

1-0

25-0

2-2

1-5

36-0

1-3

1-7

41-7

1-5

2-9

50-0

1-8

2-7

70-8

2-0

82-0

2-5

3-0

82-6

2-8

3-8

82-6

3-2

4-1

87-0

3-0

4-0

87-0

3-1

3-7

87-0

3-2

4-2

87-0

3-4

5-0

87-0

3-4

4-8

87-0

1-8

4-3

100

2-6

5-7

100

2-6

5-4

100

3-4

7-0

100

3-6

0-0

486

O. ILLMAN AND F. N, GHADIALLY

A comparison of the tumours m the brown and white hamsters showed that as
a rule the tumours m the Avhite variety contain much less pigment. Ven- often
the tumours m the bro^vn variety were so heavily melanized that no cytohmca]
details could be discerned (Fig. 2} unless the section was bleached No siioh
intensely melanized tumour was produced in any of the white hamsters. Here
most of the tumours ivere distinctly hypomelanotic and a few virtually amelanotic
(lig. 3, 4 5). However, the architecture and cytologj^ of these tumours in the
brown and w’^hite Jiamsters is essentially similar. These tumours are composed of
spindle cells and large clear polyhedral cells with an ill-defined cell boundary.
A study of the early lesions shows quite clearly that these tumours arise from the
melanocytic networks of the small spots in hamster skin. Ghadially and Barker
(1960) have already illustrated an earty melanotic tumour arising from such a
melanocytic network in the brown hamster. Fig. 6 and 7 show a similar early
lesion in a -white hamster. It can be seen that a hyqDomelanotic tumour has
formed around a group of hair follicles. The tumour is deepty placed in the dermis
and is separated by'^ a layer of connective tissue from the epidermis above the
tumour.

Epithelial tumours

Besides the melanotic tumours described above, many’- kerato-acanthomas
and some squamous cell carcinomas developed in almost all the brown and white
hamsters. In the cream varieties less than half the animals developed a few
kerato-acanthomas, and only one carcinoma emerged. Tliese tumours in the
cream liamsters started to appear approximately six Aveeks later than similar
tumours in the bromi and white varieties. Kerato-acanthomas and carcinomas
often started adjacent to the C.V. spot wdiich was destroyed as the tumours
enlarged.

DISCUSSION

Ghadially and Barker (1960) have shown that carcinogen-induced melanotic
tumours in the bromi variety of the Syrian hamster arise from a netw'ork of
melanocytes surrounding the pilosebaceous follicles in the small pigmented spots
of the sldn. Such networks are not found in mouse or rabbit sldn nor do melanotic
tumours, as a rule, arise during cutaneous carcinogenesis in these species. It can
be argued that if such net-works are a prerequisite to the production of melanotic
tumours by chemical carcinogenesis in the hamster, then a correlation between
the number of net-works and the number of melanotic tumours produced should be
demonstrable. Our results show that in the brown variety of Syrian hamster the
observable netAVorks are numerous and so are the melanotic tumours produce
during cutaneous carcinogenesis. In the cream variety the networks are seen
much less frequently and Ave haym failed to produce anj'^ melanotic tumours.
In the Avhite Amriety hoAvever no netAAwks AA^ere detected yet many melanotic
tumours Avere produced. Thus a positive correlation betAveen the number o
observable networks and the number of melanotic tumours produced Avas not
demonstrated. It seems to us that in the case of the Avhite hamster many
melanocytic networks exist but AAuth the techniques ernployed they have prm ea
undemonstrable because they contain virtuaUy no inelanm. Since ^

netAvorks are few and Avidely distributed in hamster skm, it is necessary to e-xamme

MEL.\JsOTIC TUMOURS IN' HAMSTERS

487

a large whole mount of skin to detect them, for the chance of cutting across one
of these structures in a histological section taken at random ^ '

small On the other hand, as noted earlier, a thick whole mount of skin is
suitable for the application of special staining methods for demonstrating melano-
cytes Thus it is obvious that amelanotic melanocjd-ic networks would escape
detection hv the techniques employed. The fact that inaiiy IijTome anot ic or
^^rtually amelanotic melanocjiiic networks exist m the C.\ . s]iot and that main
amelanotic and hjqiomelanotic tumours arise in the wliite variety lends sujijiort
to the idea that many small amelanotic networks of melanocytes also occur m
the rest of the skin in this variety of the SjTian hamster.

In the case of the cream A^ariety, it would appear that both the marked paiicitA
of small melanocjdiic networks in the skin and a genetic or strain resistance to the
action of the carcinogen is responsible for the failure to produce melanotic tumours,
for not only were no melanotic tumours produced but there was also a considerable
delay in the production of epithelial tumours, and a markedly poorer final tumour

jneld.

Both Horning (1958) and Ghadially and Barker (1960) have noted that when
the C.V. spot is painted with a carcinogen many melanotic tumours develoji in
the skin surrounding the C.V. spot, but virtuall}’- no such tumours develoji from
the large melanocytic networks -witliin the spot itself. Indeed only one such
tumour arising from the C.V. spot was obsen^ed by Ghadially and Barker (1960).
The present series of experiments once more demonstrates that the C.V. spot
is very resistant to carcinogenic action, for no melanotic tumours were jiroduced
from this structure.

Further, many epithelial tumours originated adjacent to the spot, which was
later destroyed as a result of enlargement and ulceration of these tumours. Thus
at times a fictitious appearance of an ulcerated carcinoma arising in a C.\k spot
was created. However, the possibilit}' that an occasional carcinoma may have
originated from the C.V. spot itself cannot be excluded, for the design of the
experiment did not permit the removal of early lesions for histology. The
possible reasons why the C.V. spot is so resistant to carcinogenic action have al-
ready been discussed by Ghadially and Barker (1960). The best e.xplanation that
can be given at the moment is that the carcinogen is probabl}’- very rapidly flushed
out of the hair follicles by the large sebaceous glands in the C.V. spot.

SUiUIARy

The skin of the brown, cream and white variety of Syrian hamsters was
painted repeateiBy with DJIBA. Many melanotic tumours were produced in
he brown and white hamsters but none in the cream variety. Most of the mejLo
tic tumours in the broum hamster produced abundant melamn but as a rurthose
,n the jariety ,-ere hypomelanotic or almost compIeMy amJLotio It k

to produce melanotTcTumoS; i„ ,1. i ° ™.‘'’ ‘ho failure

0 ,,tic netu-orhs and a genetic or strain reslreV’traS™ Kf Ja^in^r

4SS

0. IliLMAN AKD F. N. GHADIALLY

and J\lrs. A Whitaker for technical assistance. This work was supported by
grants from the University of Sheffield Medical Research Fund and the British
iifinpire Cancer Campaign.

REFERENCES

Della Porta, G., Rappaport, H., Saffiotti, U. and Shubik, p._(i956) jrcJi. Path
61, 305. ’ ■’

Ghadially, P. R.— (1959) J. Path. Bad., 77, 211.

Idem and Barker J. F.— (1960) ibid, 79, 263.

Horning, E. S. — (1958) Ciba Foundation Colloquia on EndocrinoloeiL London 12

p. 22.

Shubdc, P., Della Porta, G., Rappaport, H. and Spencer, Ivathryne. — (1956)
Cancer Res., 16, 1C31.

EXPLANATION OE PLATES

Flo. 1. — ^Bro'iTn, cream and white hamsters painted with DMBA. Melanotic tumours have
developed in the brown and white varietj’^ only. x-J.

Fig. 2.— T 3 'picnl abundantlj' melanized melanotic tumor in a brown hamster. H. and B.
X7'5.

Fig. 3. — Hj^pomelanotio tumour from a white hamster. Bulk of the tmnour cells contain
little or no melanin. Onl}' a few denselj’ pigmented ceils are scattered throughout the
tumour. H. and E. x 75.

Fig. 4. — High power view from tumour illustrated in Fig. 3, showing a few well-pigmented
cells scattered among cells containing little or no melanin. H. and E. x 6615.

Fig. 5. — An almost completel}’ amelanotic melanoma from a white hamster. In many serial
sections onij' an occasional granule of melanin could be detected. Silver staining revealed
a few more. H. and E. X 666.

Fig. 6. — ^An early lesion illustrating a hj-pomelanotic tumour arising from a meianocj’tic net-
work surrounding the pilosebaceous follicles in a white hamster. Note that the tumour is
deepl}’- placed and has no connection with the epidermis. H. & E. x 55.

Fig. 7. — High power view from tumour illustrated in Fig. 6. The cells are in close contact
with the hair follicle seen on the left of the photomicrograph. The tumour has grown as a
slieath enveloping a group of pilosebaceous follicles. H. & E. X 330.

British JotniXAL or Cancer.

Vol. XIV, Xo. .3.

British .Toitixal of Caxcf.r.

Vol. XIV. Xo. ;i.

^.•^<.- •< A ;'a ’ w> I

'•■^V'-i ' - 'si^'wif' ’Ar"

^fl'nuii ii„cl Glmdiallv.

?rof OT TOTOTHf i™ genital tiiact ol'

RATS BY CHEMICAL CARCINOGENS

CORA P. CHERRY and A. GLUCKSMAXN
Strangeways Besearch Laboratory, Cambridge

Received for ]iublication July 20,

1000

Changes in the hormonal environment affect the development of tmnoiirs
endocrine organs (Gardner, 1953; ICirschhaiim, 1957; Bielschovsky and

Torralba andMainzer (1956) and Huggins d al (1959) have shown that the ovary,
the pituitary and the adrenals influence the growth of transplanted tumours and
the induction of breast cancers by the ingestion of metliylcholanthreiic in rats.
In a previous report (Glucksmann and Cherrj-, 1958) we liave indicated that
castration reduces the incidence of vaginal sarcomas in rats after the intravaginal
application of 9, 10-dimethyl-l, 2-benzanthracene pMBA) but does not affect
the production of carcinomas of the vulva. Administration of oestrogen only
partially succeeded in increasing the tumour incidence in the vagina.

The report of the present series of experiments pursues the theme further in
an attempt to ascertain the influence of hormonal stimulation and of ablation of
endocrine organs on the formation of tumours in the female genital tract of rats.
Adrenalectomy was added to ovariectomy in the expectation of a further reduction
of tumour incidence, progesterone was given to spayed rats and tiie influence of
forced breeding was tested. The effect of repeated pelvic irradiation as a means
of castration was investigated and was also studied after surgical castration for
its influence on tumour production. Single and repeated Avhole body e.vposures
to irradiation are known to increase the rate of tumour production in various
organs (Glucksmann, Lamerton and Mayneord, 1957), to promote tumour forma-
tion in combination with chemical carcinogens and hormones (Kirschbaum, 1957)
but also to delay slightly the emergence of tumours at other sites (Lisco, Ducoff
and Baserga, 1958). Repeated whole body exposures were given to rats in addi-
tion to the intravaginal application of carcinogens to study the effect of X-ravs
on tumour formation.

Topical application of chemical carcinogens increases the incidence of tumours
of the lung (Shimkm, 1955), the ovaries (HoweU, Marchant and Orr, 1954) and
the breast (Huggins^ ah, 1959 ; Geyer, Bryant, Bleisch, Peirce and Stare 1953
It was thus thought worth while to examine the effect of additional applications

490

CORA P. CHERRY AND A. GLUCKSJIANN

MATEBIAI) and 3IETHODS

Female black-hooded rats inbred in this laboratory were used when 2 to 3
months old. The experimental animals were painted intravaginallv with a 1
per cent solution in acetone of 9, lO-dimethyl-I, 2-benzantlwaeene (DJIBA)
obtained from Messrs. L. Light & Co., ndiile the controls were painted with
acetone. Tlie application was by means of a cotton wool sivab on the end of a
galvanized wire. The vagina was stretched open by dorsal flexion of the tail
tile swab was inserted and the vagina and cervix painted bj" means of a rotarr
motion. This form of administration entailed contamination of the vulva wliich
was reduced in some experiments (Table I) b}- blotting the vulva with filter
paper immediately after the painting.

Table I. — Exjierimenial Procedures and Number of Animals

Stnto of Rnfc

Additional treatment

Number of
v-eekly paintings

Number
of rats

Remarks

I'irgin

Nil

•5* DMBA .

—

Virgin

• »» ♦

5* Acetone .

Cn.strntod surgically „

DMBA .

idem

Acetone .

oestrogen, 2 x weekly

DJIBA .

—

progesterone 2 x weekly

DMBA .

A’^ulva blotted

* ♦

Acetone .

>»

. adrenalectomy

DMBA .

' if •

Acetone .

0 X 310 r to pelvis in 20 weeks .

none

— ‘

DJtBA .

—

Acetone .

—

Virgin

• »>

DMBA .

—

AV/

Acetone .

— '

Pregnant

DMBA .

A^ulva hhtted

A^irgin

. 4 X 400 r whole body in 30 Aveeks .

DMBA .

—

. DMBA to dorsal skin 1 X Aveekly .

DJIBA .

— “

* Once iveekl^’ for 28 n-eeks and then twice weekly for f

7 weeks.

Surgical procedures. — Bilateral ovariectomy was performed under ether
anaesthesia at the age of 2 to 3 months and the painting was started 1 to 2 months
later. Bilateral adrenalectomy was performed under ether anaesthesia 3 weeks
after ovariectomy^ and painting started 1 month later. These rats were kept on
saline for the duration of the experiment.

Padiation procedures. — A pelvic field including the vagina, cervix, uterus,
ovaries and adrenals was irradiated through a heart-sliaped hole of 20 cm. in
a lead shield of 1 cm. thickness. The animals were held in position by means
of a plastic cloth clamped to a metal holder. A dose of 340 r. was given m
minutes and repeated at intervals of 4 Aveeks over a period of 20 B'eeks to a o a
dose of 2040 r in 6 exposures. Radiation factors were ; 200 KV A.-rays at lu
mA, focal skin distance 25 cm., filtration 1-0 mm. A1 and 1-0 ram. Cu,

For Avhole body exposure the animals u'ere placed in a plastic box ot 20 X -
cm. and irradiated from below. A dose of 400 r was given in 9 minutes and the
exposures repeated at intervals of 10 Aveeks over a period of 30 weeks to a tota
dose of 1600 r in 4 exposures. Radiation factors were : 200 X-rays at 10 ra ,
focal skin distance 50 cm., filtration 1-0 mm. A1 and 0-5 mm. Ou.

induction op tujiours in genital tract op rats

4 ill

Additional treatments.— Tio maintain an almost contmuons state of pregnanm
4 females ™ housed in a cage witli 1 male and the litters remoyec from (he
cage soon after birth. This arrangement gave up to 1 1 litters jicr rat in 1 0 monih.s,

though some rats proved to be less fertile (Fig. 3). ^ x r , • i- -i

A dose of 1 Hg. of oestradiol monobenzoate (Organon, Ltd.) m olive oil ua.s

iniected intramuscularly twice weekly. , , . , it

Progesterone (Organon, Ltd.) also was given twice weekly intramuscularly

in a dose of I mg. . , . , , ,

The dorsal skin region in one e.xperiment was painted with a cotton wool swan

with DMBA in the same concentration as was used for the vagina.

The experiments were performed over a period of 4 years, but there was an
overlap of several months between the different groups of experiments and witliin
each group the controls were carried out at the same time.

Histological Methods.— KminaXs were killed when definite evidence of the
presence of a vaginal tumour was available or when e.xtensive vulval tumoni-s
or other conditions made it necessarj'. The rats were inspected at least once

weekty and notes made of macroscopic lesions such as warts.

At autopsy the uterus, cervix, vagina, vulval skin and the ovaries (except in
surgical castrates) were fixed in Zenker-acetic or in the Susa fixative, dehydrated
and embedded in paraffin, sectioned at S/i. and the sections stained with haema'
toxylin-eosin, by the periodic acid-Schiff technique with or without jircvious
diastase digestion, by the Feulgen method, vith Southgate’s mucicarmine .stain,
with van Gieson’s stain or with a modified “ Azan ” stain.

Where the dorsal skin was painted, this was excised for fixation. .Adrenais
were fixed routinely and a special search rvas made for remaining adrenal ti.ssuo
in rats in which an adrenalectomj’’ had been performed.

The thickness of the uterine and vaginal stroma was measured histologicaliy
as the distance between the innermost muscle layer and the basement membrane
of the epithelium.

RESULTS

Tumour indiiction in the vagina

The majority of vaginal tumours were sarcomas arising in the subepithehai
stroma. Presarcomatous lesions and fibromas developed less frequently in the
same localization while epithelial tumours such as papillomas and carcinoma.s
occurred only rarely.

vi were cellular uith varying amounts of fibre formation, often

uith multi- or mononucleate giant cells and sometimes rrith a leiomyomatmis

Iargc.“bS ^ of ven-

492

CORA P. CHERRY AND A. GLUCKSMANN

“iddle or posterior half of the

Table 11.— Vaginal Tumours Induced by D3IBA-painthg

State

Additional

Number

Sar-

comas

of rat

treatment

risk

No.

Castrate

Nil

. 15 .

Virgin

. AAUiole body

. 20 .

Virgin

X-rays

PeU-ie X-rays

. IG .

Castrate

Oestrogen

. 1C .

Castrate

Progesterone

. 28 .

Virgin

DJIBA to skin

. 21 .

Virgin

Nil

. 23 .

Pregnant ,

Nil

. 23 .

Castrate .

Peh’ic X-rays

. 15 .

Castrate .

Adrenalectomy

8 .

Xumbtr
of rats

Presar-

comas

Fibro-

mas

Carci-

nomas

Hearts

with

All vaginal
tumours tumours

No.

'• %

1 —
No

'• %

0 ,

. 3

. 6

0 .

4 .

13 .

100

7 .

12 .

100

Table II gives the incidence of tumours after the different treatments. None
of the acetone-painted controls (Table I) developed anj’^ tumours in the vagina
or the vulva. Animals “ at risk ” survived the first painting for at least 150 days.
The incidence of tumours ranged from 20 to 100 per cent and tlrree levels can be
distinguished. At the lowest (20-30 per cent) were the rats painted after surgical
castration or castration bj’^ repeated whole body irradiation. At an intermediate
level of tumour incidence (43-50 per cent) Avere (a) virgin rats subjected to pelvic
irradiation, (b) surgical castrates given oestrogen, (c) surgical castrates given
progesterone and (d) Aurgin rats AAdiose dorsal skin Avas painted AAuth DMBA.
The incidence of sarcomas of the Amgina is the same in the Ioav and intermediate
group, but in the latter fibromas and presarcomatous lesions are found in addition
to sarcomas and raised the total tumour incidence. The highest level of tumour
incidence AA’as achieAmd b3’- (a) Aurgin rats, (b) pregnant rats, (c) surgical castrates
subjected to repeated pehdc irradiation and (d) surgical castrates AA-ith bilateral
adrenaleetom}'. In this group onl 3 ^ AA’ere found papillomas and carcinomas of
the Arngina (Table III), giAung rise to multiple tumours (carcinoma or Avart plus
sarcoma) in the same rat. In this group the incidence of tumours exceeded that
of tumour bearing rats (Table II) Avhile the tAAm figures are identical at theloAver

levels of tumour incidence. ■ • f

The difference in total tumour induction betAveen the tliree levels rs signihcant
at the 95 per cent confidence level. The high and intermediate tumour groups

Table 111.— Type of Vaginal Tumoiirs at Three Different Incidence Levels

Incidence Sarcomas
leA'el %

Low . 26 Jz

Intermediate . 37 d: S' 4

High . 74 ±5-3

Presarcomas

fibromas

10 ±3-5 .

4 ±2-4 .

Carcinomas

papillomas

o/-

13 ±4-0

All

tumours

0 /

20 ± 7-4
47 ± 5-5
01 ± 3-4

INDUCTION OF TUMOURS IN (SENITAU lUACI OI KAIS

.in:{

also differ significantly in the incidence of sarcomas ^liile t here is^ no
Serence in this respect beUvecn the low and the mtermed.ate lei el I he r s
io a significant fall in tlie proportion of

to sarcomas in the progression from the intermediate to the high IiAtl . '-i ■ '
are 9 non-malignant lesions and 29 sarcomas in the

3 non-malignant lesions and oS sarcomas in the high incidence g l ( •

Time ranges in tumour development

Since animals were killed at the first definite indication of vaginal tnmonrs
or when large vulval growths or other conditions made it necessary, the true
induction time could not be determined. As the tnmonrs varied in size when first
discovered, no reliable estimate of the period of growth can be given. 1-or the
same experiment the average “ surrdval ” time of rats with .sarcoma.s and those
without was not statistically different except for surgical castrates gi\en jieh ic
irradiation (Table IV). There are marked differences, however, between the

Table XV. — Average Survival Period for Pats with arid Without Vaginal Sarcomas

After DMBA Painting

Survival in

days of rats

State
of rat
Castrate
Virgin

-Additional

treatment

NT7

MTioIe body X-rays

Witli

sarcoma

328 - 1 - 4C- 1
290 ± 19-7

IVitliout

sarcoma

373 -J- 17-0 .
310 ± lo-r, .

Difference
45 -I- 49- 1
14 ± 25-0

Virgin

Castrate

Virgin

Pelvic X-raj'S
Oestrogen
Progesterone
, DMB.A to .skin

248 i 9-4
341 ± 33-3
260 ± 25-1
270 ± 3-9

239 -t- 9-9 .

324 -I- 14-8 .
298 4 - 38-1 .
257 in-2 .

9 ± 13-0
17 i 36-5
38 ± 45-0
13 i 11-8

Virgin

Pregnant

Castrate

AT7

Xt7

. Pelvic X-rays
Adrenalectomy

337 4- 8 0
246 -t- 9-9
217 ± 7-2
295 ± 38-8

311 -t- IG-4 .
250 ± 21-1 .
184-1- 2-0 .

227 4 - 42-5 .

26 -i- 18-2
4 ± 23-4
33 i 7-.5
68 i 57 ■ a

various experimental groups (Fig. 1). In the high tumour incidence group the
speed of tumour development was the fastest in 3 of its 4 members, while the fourth
(virgins painted with DMBA) lagged behind the 3 fastest members of the intermedi-
ate group. The speed of tumour development -was slowest in the low incidence

™ intermediate group even some of the oldest
animals failed to form tumours.

speed of tumour development at the three levels is considered
(Fig. 2), there IS a clear tendency for a shift to the right suggesting a correlation
between speed and incidence of tumour formation. It remains obsfure however
Q-t the same level of tumour incidence the sneerl '

CKperiment.1 series. Seasonal variata a« 00 ^ 0 ^^
ence, since the experiments overlapped in time. ^

Sarcomas and presarcomatous lesions appeared at abmit tVio o t

the e.xperiment with forced breeding fFii^ 3 I fL + “

rats. Of the three animals that failed to prodnStamo^^Ld 'tSS

494

CORA P. CHERRY AND A. GLUCKSMANN

Fio. 1. — Cwnultitivo percentage of all vaginal tumours for different treatments additional to
painting u'ith D3IBA : (1) castration, (2) whole body X-rays, (3) pelvic X-rays, (-J) castration
plus oestrogen administration, (5) castration plus progesterone treatment, (6) painting of
dorsal skin with DlilBA, (7) virgin rats, (8) forced breeding. (0) castration plus repeated pelvic
X radiation, (10) castration plus adrenalectomy.

Fig. 2. — Cumulative percentage

of all vaginal tumours in high (A), intermediate (B) and low (C)
tumour incidence group-

495

IKDUCTIOK OF TUMOURS IN GENITAL TRACT 01' RATS

tion reduced the incidence of veginal “ f„7vIV-™' "troma in

lectomy while the tumour production was greatlj increased, u . >

Fig. 3. — ^Number of litters and tumour incidence in individual rats painted ivith U.MBA and

subjected to forced breeding,

O = no tumour, -i- = sarcoma of the vagina, • = pre.snrcomatous lesion.

painting of the dorsal skin did not reduce the thickness of the uterine and vagina!
stroma, but decreased tumorigenesis in the vagina.

The secretory activity of the endometrial epithelium and its glands ivas re-
duced by castration, restored by oestrogen or progesterone treatment and less
regularly by adrenalectomy. The last procedure failed to aifect the atrophy of
the uterine stroma and progesterone had only a slight effect on it. Thus even nor-
mal tissues, i.e. stroma and epithelial components of the uterus, react in a different
manner to hormonal stimulation and neither shows a consistent correlation with
the liabibty to tumorigenesis.

The same applies to the vaginal stroma which though, reduced by castration
did not show a correlation of its width mtb tumour formation (Table V). This
finding confirms previous observations (Glucksmann and Cherry, 1958) and the
inclusion that the vaginal stroma responds to oestrogenic stimulation but to
Di\IBA only locally and not generally,

The squamous hyperplasia of the vaginal epithelium induced by DMBA
painting was diminished by surgical spaying and by castration ivith whole bodv

S partially the hyperplasia lost on castration. In the nreenant r-it
tate of hjqierplasia and the type of epithelium varied irith the stag! of preg-

496

CORA P. CHERRY AND A. GLUCKSMANN

Table V .—Incidence of Vaginal Tumours and Width of Uterine and
Uroma in miBA-treated and Control Rats

Vaginal

State

Additional

Tumo

of rat

treatment

Castrate

Nit

. 20

Tirgin

. Wliolo body X-rays

. 30

t^rgm

. Pelvic X-rays

. .50

Castrate

. Oestrogen

. 50

Castrate

Progesterone

. 47

Virgin

DMBA to skin

. 43

Virgin

Nit

. 78

Pregnant

Nil

. 100

Castrate

Pelvic X-rays

. 93

Castrate

. Adrenalectomy

. 100

Width of uterine strom a Width of vaginal stroma

DMBA

painted

32 ± 4-4

43 ±8-3

Control

32 ±3-7 ,

DMBA

painted

■ 15 ±2-6

■ 17 + 1-9

Control

14 ±2-0

46 ± 15-7
83 ± 7-2

39 ± 4-04
70 ± 6-7

32 ±5-2 .

39±4-l .

21 ± 2-6

23 ± 2-6

19 + 2-1

25 ± 2-3

19 ±3-3

20 ± 2-2

81 ± 8-8

80 ± lO-O .

28 ± 3-9

29 + M

35 ± 6-7

27 ± 3-1

30 ±3-2 !

28 ±6-3 .

21 ± 3-1

16 ± 0-5

17 ^1-8

18 ± 1-4

nancj'’ . at and around term, tlie epithelium it^as formed by mucin secreting, liigh
columnar cells while in the earl3r phases of pregnancy hj^erplastic squamous
epithelium was present. Irrespective of the stage of pregnancy the epithelium
overlying a sarcoma was always of the squamous tjqie.

The degree of h3q)erplasia of the Arnginal epithelium was greatest near epithelial
tumours and since these occurred onJ3’- in the highest incidence group, there was
some correlation het\reen the h3fperplasia of the vaginal epitjielium and the
tendenc}’’ to form epithelial tumours. This applied, however, to the localized
rather than generalized epithelial h3q3erplasia of the vagina. A relatively liigh
epithelium occurred in castrates after progesterone treatment and in virgins
treated with painting of the dorsal skin, but in neither of these groups were epithe-
lial tumours found. Progesterone also stimulated the cervical epithelium, and
the high columnar cells at this site contrasted strongly witJi tlie squamous epithe-
lium of the vagina.

Adrenal remnants were found in 7 of the S tumour-hearing adrenalectomized
rats. They consisted of abnormal looking cortical tissue Ijdng in the fat close
to the capsule of the kidne3L The functional activit}'' of these structures could not
be assessed, but tlie rats had to be maintained on saline solution and lost weight
immediately on being put on tap ivater. It cannot be decided whether tliese
remnants are due to the regeneration of cortical tissue left behind at operation or
to the h3^pertrophy of small foci of accessor3'^ cortical tissue in the rats. These
remnants u^ere also found in the controls treated with acetone and tended to
increase in size with time after adrenalectomy.

The ovaries of rats in u'hich the dorsal skin as well as the genital tract were
painted with DMBA showed no abnormalities except for one animal in which
the ovaries ivere largefy replaced b3^ C3'sts. This rat had no vaginal tumours,
but a carcinoma and a sarcoma in the dorsal skin and a papilloma on the m la.

Tumour induction in the vulva

All but one of the 145 vulval tumours were derived from the epithelium and
71 per cent were squamous cell carcinomas uiiile 28 per cent were
basal-celled papillomas. Only one sarcoma wms found at this *

skin showed very marked h3q)erplastic changes after DMBA painting ( uc 's

407

EsDUCTION of tumours IX GENITAL TRACT’ OF RATS

and Cherr 5 % 1958) and the degree of h>Ter])!asia as well as the incidence of tuinoiirs
.vas ind^endent of castration, hormonal treatments and

considerably reduced though not entirely suppressed by the blottii g of the il a
immediately after painting (Table M.). Tumour induction in the vuha thus

Table VI. — Tumours of the Vulva Induced by DMBA-jmintiiuj

Number

State

Additional

, —

^ »

( —

of rat

treatment

risk

No.

Castrate

Kit

. 11

78 .

Virgin

Vniole body X-rays .

. 17

Virgin

Pelvic X-rays

. 14

88 .

Castrate

Oestrogen

. 9

Castrate

Progesterone

Virgin

DMB.A to skin

. 16

Virgin

iViV

. 15

Pregnant

A’i7

Castrate

Pelvic X-rays

. 11

Castrate

Adrenalectomy

. 3

Pnpil-

lotnns

ir>

18 .

in .

20 .
43 .
27 .
25 .

Snr-

coinns

No.

All

tumours Number of
weekly
No, % piiintiufr^

12 80
20 100

0 . 10 100 .

0 . 13 81 .

0 . 7 25 .

5 . 21 100 .

02 .
05 .
100 .
02 .

1-5

= Vulva blotted with filter paper after painting of vagina.

appears to be independent of hormonal state but to varj" with dosage of the carcino-
gen at certain dosage levels. Single applications per week induced as many
tumomrs as two applications per week. Only blotting after single weekly applica-
tions reduced the tumour incidence.

The speed of tumour formation varied markedly between the different e.vperi-
ments as seen in Fig. 4, in which the cumulative percentage of tumours is plotted
against the time of histological confirmation. Table ITl gives the interval be-

Table VII. — Incidence of Tumours at the Vulva and Time to Afpearance of First
^Yart After DM BA -painting of the Vagina

State Additional Tumours First wart

of rat
Castrate
Virgin

Virgin

Castrate

Virgin

Pregnant

Castrate

treatment

Nil

VTiole body X-rays .

100

. Pelvic X-rays

100

Oestrogen

. Progesterone

25* .

. DMBA to skin

100

Nil

65*

. Pelvic X-rays

100

. Adrenalectomy

62* .

* ~ Mulva blotted after vaginal paintin;

da vs
205 (248)
154

135

136
193
II2

203

120

135

269

but in one experiment on snrgiejUy castrated ratra'b” ‘°!“f ">■ “nlinimtion,
on histological examination 20 days before the fir«i- tumour was found

The basal cell tumours could not b^ snSted mn ^ “f^rf copic warts appeared,
rather than outwards. ^ macroscopicaUy as they grew inwards

498

CORA P. CHERRY AND A. CLUCKSiAIANN

vulva. Since in pregnant rats the first warts appeared early in spite of the blotting
after the painting, it is doubtful Avhether dosage of the carcinogen hastened the
formation of tumours. On the other hand the proportion of papillomas to car-
cinomas was significantly greater Avhen the vulva was blotted (17 warts of 27
tumours or 63 per cent) than when this procedme rr^as omitted (24 warts of 1 17
tumours or 21 per cent). Thus the degree of malignancy as rvell as the total
incidence of tumours decreases irdth the reduction of contact rvith the carcinogen,
but the induction time for the first rvarts is not lengthened.

DISCUSSION

The tumour incidence in the vuhm and vagina is the same ivhether the D5IBA
is applied once or twice a iveek and thus is independent of dose at this level o
carcinogenic stimulation. The incidence particularly of malignant Auilval tumours
is decreased by blotting after single weekly applications of DMBA and at this dose
leiml tumour development of the vulva is obviously dependent on dose. It is
noteworthy that in the vulva as in the vagina the increase in total tumour inci-

INDUCTION OF TUMOURS IN GENITAL TRACT OF RATS

4on

deuce is accompenied by an increase in tlie proportion of malignant lesions

emic factors tested by the additional treatments rSienJ

earinarbut not of vulval tumours and thus ue have to cons dcr the me picnt
Loplastic tissue of the vagina as susceptible to ^stenne and in P“;‘.en ar to
hormonal action while the vulval skin is not. Tlie response of the tu nour-
forming tissue of the vagina to hormones differs from that of the normal stroma
of the vagina from which it is derived and also from tliat of tlie uterus of tlic same
animal Thus the tumour incidence does not var}' with the width of tiie vaginal
or uterine stroma (Table V) which responds to oestrogenic treatment, f^imdarly
Hudgins ei al. (1956) found a difference in the response of a transjilanted iihro-
adenoma and that of the normal breast to hormonal stimulation in ovaricctomised
rats. Progesterone accelerated the growth of the tumour but not that of the
breast. Oestrogen stimulated the growth of the breast irrespective of dose and
the growth of the tumour in small doses, but retarded it in large doses. In our
experiments a dose of oestrogen sufficient to restore fully the width of the uterine
and vaginal stroma of castrates failed to restore the tumour incidence though it
increased it. The dose of oestrogen used was in the lower dose range which in
Huggins’s experiments still had a stimulating effect on the fibroadenoma and
promoted tumour induction in the breast (Huggins et al., 1959). The effect of
oestrogen on tumour production in our experiments was of the same order as
that of progesterone but in Huggins et al.’s experiments much larger doses of
progesterone promoted the induction of breast tumours verj’ much. The difference
in the results may thus be attributable to dosage, rather than to a difference in
reactivity of the breast and the vagina.

Surgical castration and castration by repeated whole body irradiation greatly
reduced the tumour incidence in the vagina but did not completely abolish it
(Table III, low level). similar effect of surgical castration on the induction
of breast tumours has been reported by Huggins et al. (1959). The administration
to ovariectomised rats of progesterone or oestrogen induced presarcomatous
and fibromatous lesions and did not greatly increase the incidence of vaginal
sarcomas (Table III, intermediate level). Tumour incidence was also reduced
by the slow castration of rats through repeated pelvic irradiation and by the paint-
ing of the dorsal skin with DilBA. The latter procedure produced no ovarian
tumours and ovarian cysts occurred in only one rat which failed to develop a
vaginal tumour. The measurements on the vaginal and uterine stroma do not
suggest a deficiency in the oestrogenic stimulation in these rats. In this respect
the experimental results resemble closely those obtained with oestroo-en administra-
tion to ovariectomised rats. It is significant that in this intermediate group of
tumour incidence the increase is due mainly to the appearance of presarcomatous
and fibromatous lesions whde in the high incidence group the increase is due
to sarcomas and the non-mahgnant lesions become rare (Table III, high level)
incidence ^oup belong the virgin and the pregnant rats\nd also

he surgical castrates with adrenalectomy and vith repeated pelvic irradiation

that in and pregnant rats is not unexpected but

500

CORA P. CHERRY AND A. GLUCKSJIANR'

induction of vaginal sarcomas. The remarkable promotion of tumorigenesis mav
be due to the persistence of possibly active adreno-cortical remains. ^Even then
sorne abnormality m the function of the adrenal must be assumed. It is feasible
that an abnormal function of the adrenals is also responsible for the high tumour
incidence in spayed rats receiving X-rays over a pelvic field including the adrenals.
Ovariectomy may be followed by the hyperplasia of the adrenal cortex and even
tumour formation (Bielschowsky and Horning, 1958) in response to high levels of
pituitary gonadotrophin after castration. Irradiation of the adrenals in such a
state but not in the intact animal may produce an abnormal secretion able to
promote tumour formation in the vagina but vdthout efi’ect on the vldth of the
uterine and vaginal stroma.

The complexity of the hormonal interactions is also illustrated by the e.xperi-
inents of Shay, Harris and Gruenstein (1952) in ivhich breast tumours were
induced in male and female rats bj'^ the administration of methjdcholanthrene
bj'- stomach tube. Castration of females greatly reduced the tumour incidence
but testosterone and progesterone only slightly lowered the tumour incidence in
intact females. On the other hand oestrogen treatment much increased the
tumour incidence in intact and castrated males. The interplay of various endo-
crines is also brought out b}'^ Huggins et al. (1956) who also show'ed that h}'pophy-
sectomy inhibits the growth of the transplantable fibroadenoma, that oestrogen
alone had hardly any effect on the tumour growth in such animals, that the com-
bination of oestrogen and progesterone was more effective and that this effect was
still further increased by the addition of lactogenic hormone or growth hormone.
In our experiments castration decreases the tumoim incidence and nidtli of the
vaginal and uterine stroma ; the administration of oestrogen increases the vddth
of the uterine and vaginal stroma, but only slightly enhances the tumour formation
in the vagina ; adrenalectomj'^ and pelvic irradiation significantly increase the
incidence of tumours without stimulating the growth of the uterine and vaginal
stroma.

It is quite obvious that the endocrine and systemic influences discussed here are
not of generalized nature, i.e. they do not affect all tissues alike but exert their
action only on specific target tissues. Whether they act as sensitizers to the
initiating action of DMBA or promote the growth of changed cells, cannot be
decided. It is noteworthy, however, that the non-mah'gnant forms appear first
at the intermediate level of tumour induction and that only at the highest level
of induction do the epithelial tumours appear in addition to the increased number
and proportion of sarcomas. Though on the average the speed of tumour
ation is also increased in the same direction (Fig. 2), the differences in indirndual
experiments (Fig. 1) sho-w that the rate and speed of tumour induction are not

necessarily closely linked with one another. _ , ■ i

The absence of a correlation between the width of the uterine and vagina
stroma and the tumorigenesis shows that the systemic itifluences cannot be con-
sidered as general promoters of grondh in the form of mitotic stimuh. le ac
that castration decreases the tumour incidence and oestrogen slightly increases
it is evidence against the hypothesis of Jackson and Robson (1957) that oestroge i
hormones compete with carcinogens for the specific growth receptors. ®
rather that specific levels of reactivity to DMBA in the tissues of the ^

and of their stimulation by as yet undefined endocrine agents have to be P®
to explain the differential behaviour of vaginal stroma and vaginal epithelium

501

INDUCTION or TUMOUKS IN GENITAL 'IRACl 01 UAIS

in tumour formation. Tlie vulva shows sucli a liigli rcspomsivcness to DSUiA tliat
an influence of systemic factors has been obscured if it exists at all.

SUMMARY

Ovariectomy reduced the incidence of vaginal tumours after intravaginal
apphcation of DMBA, and administration of oestrogen or of progesterone raised
the incidence of tumours only slightlJ^

Repeated whole body exposures to X-rays also lowered the rate ol tumour
incidence after painting and so to a lesser extent did repeated pelvic irradiation
of virgin rats and the apphcation of DMBA to an additional dors.il .skin region.

In surgical castrates adrenalectomy or repeated pelvie irradiation restored
the level of tumour incidence to that of intact and pregnant rats.

Three levels of vaginal tumour incidence are found and the distribution of
tumour t^Ties and the length of the average induction time varied u'ith the level :
at the lowest level there are only sarcomas, at the intermediate level fibromas and
presarcomatous lesions are found in addition to the sarcomas while at the highest
level the incidence of sarcomas is increased and epithelial tumours appear.

Tumour induction in the vulva is not affected by castration, radiation or
hormone treatment but varies at certain dose levels with the dose of the carcinogen.

The authors have pleasure in acknowledging their gratitude to Dr. H. B. Fell,
F.R.S. for her constructive criticism of the manuscript, to Mr. G. C. Lcnney for
technical assistance, to Mr. H. G. Hignall for irradiating and Mr. G. W. Stebbings
for looking after the animals.

This work was carried out under a grant from the British Empire Cancer
Campaign.

REFERENCES

Bielschowsky, F. axd Hornxng, E. S. — (1958) Bril. med. Bull., 14, 106.

Gaedxer, W. U. — (1953) “ Hormonal aspects of experimental tumorigencsis ”. In
‘ Advances in Cancer Research ’, Vol. 1. New York (Academic Press), p. 173.
Geyer, R. P., Bryant, J. E. Bleisch, V. R., PEmcE, E. M. and Stare. F. J —119531
Cancer Res., 13, 503.

Glucksmann, A. AND Oherry, C. P.— (1958) Brit. J. Cancer, 12, 32.

Idem, Lamerton, L. F, and Mayneord, W. V. — (1957) “Carcinogenic effects of radia-
tion Jw Raven, R.W.,‘ Cancer’, Vol. 1,497. London (Buttenvorth & Co )
Griffin, A. C^Rinfret, A P and Corsigilia, V. F. (1953) Cancer Res., 13, 77.

^ Re^.,'i K.-(1953) Proc. Amer. Ass. Cancer

Howell, J. S^ JIarchant, J. and Orr, J. W.— (1954) Brit. J. Cancer 8 635
Hugg^s, G., Briz^elli, G. and Sutton, H.— (1959) J. exp. Med 109 4 ‘

Idem, Torralba, Y. and Mainzer, K.— (1956) Ibid., 104 525 ’’ ’ “ '

loi.

Muhlbock 0.— G9o6) “ The hormonal genesis of mammary- cancer ” In ‘ M

SinMKiN, M. B. flOaSi “ Pi,i,v,„ ’ i Gawcer /n.sG, 13 307 .

“ Advances in Cancer Researc^’^ Vor™°'^Nei\^y®1F^A™T*'‘- ”•

Slawikowsky, G. j. M.-(1960) Cancer Res.', 20, m ^ Press), p. 223.

502

REDUCTION OF MAMIARY CARCINOMA AND ADENOJIA IN CHf
BREEDERS AFTER LATE OVARIECTOMY *

B. D. PULLINGER

From the Cancer Research Deparhnent, Royal Beatson Memorial Hospital, Glasgow
Kcceived for publication July 13, 1960

The earliest reports of reduction in mammary carcinoma after ovariectomy
ivere made in inbred virgin female mice at various ages (Lathrop and Loeb, 1916 ;
Loeb, 1919 ; Cori, 1926, 1927) and in former DBA breeders (Murray, 1928, 1932^
1936). These experiments preceded the discovery of the msunmarj tumour
agent by Bittner in 1936. On account of tlie liigh spontaneous incidence of this
tumour in tlie mouse strains used at that time and the evidence obtained since
then of infection of DBA mice u'ith the agent, it is probable that all reported
reductions occurrred in infected stocks. Later observations, which liave con-
firmed earlier results, have been made with C 3 H mice known to carry the agent
(Shimkinand Wyman, 1945 ; Pilgrim, 1957). It seemed probable that a reduction
would be found also in the absence of biologically detectable agent. Mammarjf
carcinoma urns too rare in the RHIf strain without the agent to judge whether
ovariectomy would prevent it but adenoma incidence was reduced 15-fold
(Pullinger, 1955). Up to and since that time altogether 70 former Rlllf breeders
have been ovariectomised rvithout finding a mammarj^ carcinoma subsequently
but from the data of Pullinger and Iversen (Table I, 1960) this total is insufficient
to judge whether or not a reduction has been achieved.

The CgHf substrain bred by Heston and his colleagues has an overall incidence
of 22 per cent of mammary carcinoma. Exhaustive biological tests failed to
reveal the presence of the mammary tumour agent (Heston et al., 1950 ; Heston
and Deringer, 1952, 1953; Heston, Deringer and Dunn, 1956; Heston, 1958).
Through the kindness of Dr. W. E. Heston, progeny of CgHf/He mice have been
bred in this laboratory for the present experiments. Among 108 breeding
females 28 or 25-9 per cent, developed mammary carcinoma (Pullinger and
Iversen, 1960). No biological evidence of agent Avas found (Pullinger, 1960).
The fact that after gonadectomy at any age mice of CgH ancestry, in common
with that of some other strains, are liable to develop adrenal cortical hyperplasia
and carcinoma, often udth biological evidence of secretion of oestrogen (Smith,
1948) might have made it impossible to test the effect on incidence of manimary
tumours of lack of oestrogen from knorvn sources unless adrenalectomy had a so
been done. The risk that actively secreting adrenal cortical tumours would arise
in these mice after ovariectomy’' at puberty proved to be small (Pullinger, 1 )■

The older the mice when ovariectomised the less this risk is seen to be, thoug 1 1
is never wholly absent in strains with the tendency. - f i q

In the present experiments on 50 former CgHf breeders a reduction ol i to m
fold in mammary carcinoma was observed after late ovariectomy. One mammary
carcinoma arose among 50 ovariectomised mothers. This mouse la a s
microscopic invasive adrenal cortical carcinoma with evidence m e ,

oestrogen secretion. One macroscopic non-secreting adrenal tumour o

MAMMARY TUMOURS IN MICE AFTER OVARIECTOMY

r,03

JIATERIALS AND METHODS

The origin, tumour rates and management of tlic CaHf colony and treatment
of tissues have been recorded (Pullinger and Ivcrsen, 19G0). Animals used in the
present experiments were derived from surplus litters of the breeding colony.
Temales were mated either with their brothers, in which case their ])rccisc ancestry
was knovm, or they were collected at approximately the same ages in batches of
6 or 7 and mated at random. The experiment required two groups of breeders,
one segregated only, the second segregated and ovariectomised at tlie same age.
Further experiments, to be recorded later but begun concurrently, included sub-
stitutions of ovarian hormones after ovariectomy. Whole families of line-bred or
batches of randomly mated females were allotted in turn to these several groujxs.
No deliberate selection of mice was made for the various groups ajxirt from the
exclusion of a few mothers that had no more than one or two litters. None of
the latter developed mammarj’’ carcinoma. Because no selection was made, the
number of females having the same number of pregnancies are not e.xactly matched
and because breeding was curtailed by segregation or by ovariectomy the most
fertile females, presumably capable of bearing 9 to 12 litters, were prevented from
having more than 8.

The age chosen for segregation or for ovariectomy determined the actual
number of pregnancies of indi\ddual mice. Originally this was 8 to 9 months in
order that the experiment might start before the first mammary carcinomas wore
expected. Some have been reported bj’’ Heston et al. (1950) al 9 months of age
but as none had yet been seen in our colony before 12-4 month?, seirreo-ation and
ovariectomy were eventually postponed to 9 to 11 months in all^but 8 of the
females in Tables I and II.

Table I. Incidence of ^letTUTnavy Oexvcinoinei A. 7 nony Hefevciice ond
Ovariectomised Former C^Hf breeders with Number of Litters

Number

litters

8
9

Totals
Per cent .

.Segregated breeders

Reference group

Number

mothers

13
9

14
9

4
8
0

5
9

Number

with

eareinoma

3
2
2

4
2

5
0
1
2
1

Intact

Number

mothers

26-3

* Mouse with adrenal cortical

13-8

carcinoma.

Ovariectomised

Number

with

carcinoma

Number

mothers

.50

Number

with

carcinoma

1 *

504

B. D. PULLINGER

Table l\.~lncidence of Mamma^ Carcinoma in Meference Groups and in
Ovaj leclomtsed Former C^Hf breeders with Survival Ages

Reference group

Survival

Number

ago in

with

montlis*

motheraf

caroinoma

Totals .

For cent .

26-3

Segregated breeders

Intact

Number Number
of with

mothers carcinoma
0 0
0 0

0 0

t 0

2 0

3 1

3 0

2 I

2 0

4 0

2 1

1 0

3 0

4 2

4 0

5 0

0 0

36 5

Ovariectomised

t ^

Number Number
of with

mothers carcinoma
0 0
0 0

0 0

I 0

0 0

1 0

2 0

4 0

1 1

1 0

5 0

4 0

6 0

8 0

6 0

3 0

2 0

I 0

0 0

13-8

* Age at deatli or appearance of mammary tumour,
t From Pullinger and Iversen (1960) less 17 which were segregated.

Bilateral ovariectomj’' was done by the abdominal route using hromethol
anaesthesia. The mice were allowed to live until natural death or tumours were
found or the3r tvere unable to feed. All ovariectomised and segregated mice were
seen daily and palpated weekly. After death many adrenal glands and all that
were enlarged or nodular were examined microscopically as also were tumours of
this and other sites. All 10 nipple regions from samples of mammary glands from
both groups were fixed for bulk-staining and for counts of adenomatous nodules.

RESULTS

One mammary carcinoma arose among 50 ovariectomised breeders (Tables I,
II and IV). It was a cj'^stic adenoacanthoma in a fourth left nipple region. No
trace of ovarian tissue was found. Both adrenal glands were slightly enlarged and
nodular. On microscopic examination hyperplasia and wdespread infiltration of
both capsules and of extracapsular fat by large, vacuolated type B cells of Wooliej
and Little (1945) were found. Evidence of oestrogen secretion, probably from
this latter source, was seen in the uterus, which, at 13 months after ovariectomy
was hypertrophic. The horns w'ere 2-5 mm. and the common uterus 3-0 mm. m

MA:iIMARY TUMOURS IN MICE AFTER OVARIECTOMY

^^^dth. The epithelium was of mucous-secreting tyiie with foci of i)olymorph()-
nuclear cells. Epithelial cells were found in mitosis. In all other females at tins
age whether ovariectomised or intact, the remains of genital organs were atrojihic.
The nipple regions of this mouse were completely involuted apart from the j^rcsence
of 6 adenomas (included in Table III). A single female with a mammary carci-
noma among 50 ovariectomised breeders reveals a reduction in incidence in

Table III. — Incidence, of Adenomn in Samples of Reference Grotijm
and in Ovariectomised Former C^Hf breeders

Number of Number

breeders with

exnmincd ndenoina

Reference group . 37 . 31

Segregated only . 13 . 12

Ovariectomised . . 23 . 11

Total

ndenoma.s

.344

233

Average number
of adenomas jier
mouse nffected
17-3
HI-4
3-0

comparison tvith 24 females with these tumours among !)1 normally bred or 5
among 36 that tvere segregated after breeding. The degree of this reduction must
be judged in relation to parity and survival age of the breeders. Instead of
comparing averages, all females that had no more than 8 pregnancies may be
considered. Out of 81 normally bred mothers from the reference group taken
from PuUinger and Iversen (1960), 20 developed mammarj’ carcinoma and of 36
segregated there were o compared with 1 only among the 50 ovariectomised (Table I).
The difference in incidence bet^veen the reference group and those segregated only
is not statistically significant, (y^ = 1-15). The differences between both
segregated and reference groups and those ovariectomised are significant (y- =
25-4) and amount to reductions in incidence of 7 and 13 times.

In comparing survival ages, only those animals which lived to at least one
month after operation or segregation are included in these results. None of 5
that were ovariectomised and died earlier developed mammarj’^ carcinoma. There
is no significant difference in survival time between the three groups (Table II and
Eig. 1).

Adrenal glands of 28 ovariectomised . breeders in addition to the one just
referred to were examined microscopically.- Results are summarised in Table IV

Table IV. Adrenal Cortical Changes and Mammary Tumours
in Ovariectomised Former CJHf breeders

Cortical B cells
Present only

Simple hyperplasia
Invasion of capsule
Gross carcinoma

{

Number of
former
breeders
17
4

4
1

Number with ;

^lammary
Adenoma carcinoma

r»

- Indicates no obser\-ations made.

T^Tie R, potential oestrofren-seerptinfr

both of 21 pairs ; in 3 pairs thev wpvp b ’ ^ iR the cortices of one or

506

B. D. BULLINGEE

tion of oestrogen. One gross adrenal carcinoma was found at 3 ^ mnnfhc . .3

(hyperpiastic nodules) were found in II
f these fema]^ a 5-foid reduction compared Avith intact breeders (Tabie III)
breeders a 15-fold reduction of adenomatous nodules was
a ei ate ovariectomy (Pullinger, 1955). Though niammarj^ carcinoma

'>v

Flo. I . — Percentage of sun'ivors at ages given in months.

# = The reference group of Pullinger and Iversen (1900) less IT.
X = The group of intact segregated former breeders.

O = The group of ovariectomised former breeders.

undoubted]}’’ progresses tlirough a stage wdiich, at the present time, is morpho-
logically indistinguishable from adenoma, it is uncertain, in the absence of Bittner’s
mammary tumour agent, irhat are the potentialities of most of these adenomas.
Thirty-six nodules from Blllf females lacking evidence of agent failed to
■when grafted subcutaneously or intraperitoneally into young homozygous virgin
or breeding females. These hosts lived for further periods of 10 to 18 months
thus allowdng time and a continued normal hormonal environment to favour their
proliferation and change to malignancy. All palpable carcinomas in this strain
had proved to be transplantable in subcutaneous tissues of homozygous females
and males. The conclusion was reached that the potentialities of the nodules was
limited (Pullinger, 1954). However wdien the mammary tumour agent was
present, Broivning (1948) grew’ several small nodules in the anterior cham ers
of eyes of homologous hosts. Recently, by regrafting nodules from agent-

MAMMARY TUMOURS IN MICE AFTER OVARIECTOMY .)0/

infected C,H mice into mammarj^ fat pads from which normal j)re-existmg
mammary epithelium had been excised, De Ome ct al. (1959) grew maminarv
carcinoma from nodule transplants that had failed to grow in subcutaneous tissue.
The potentialities of agent-free nodules now need retesting by grafting into fat
pads bj^ this technique.

Of tumours of other sites in the 50 ovariectomised breeders there were (i
hepatomas, 6 pulmonarj' adenomas and 1 epithelioma of skin in a mouse which
had also a subcutaneous sarcoma. Macroscopic hmiphoblastoma and reticulum-
celled tumours were found in 4. Polyoma antibody was found in 7 out of 1 0 of
a sample of the C 3 Hf colony.

StnMJIARY

A reduction in mammarj’- carcinoma occurred after ovariectomj' in a grouji of
50 former breeding females. This varied from 7 to 13 times in comparison with
2 reference groups, one of which was segregated when the mothers were ovari-
ectomised and the second group was bred normallj'. The incidence of adenomas
was reduced 5 times.

The one mammarj’’ carcinoma wliich arose in the group of 50 ovariectomisefl
breeders was associated vith an adrenal cortical carcinoma and evidence in the
uterus of endogenous oestrogen secretion.

I am indebted to Dr. S. Iversen for Fig. 1.

REFERENCES

Bittner, J. J.— (1936) Science, 84, 162.

Browning, H. C. — (1948) J. -noL Cancer Inst., 8, 173.

CoRi, C. F.— (1926) J. Cancer Res., 10, 265.— (1927) ./. exp. 3Ied., 45, 983.

De Ome, K. B., Fauekin, L. J., Bern, H. A. and Blair, P. B. (1959) Cancer Res., 19,
ol5» ^ *

Heston, W. E.— (1958) Ann. N.Y. Acad. Sci., 71, 931.

Idem DraiNGE^ M.^K.— (1952) ./. nat. Cancer Inst., 13, 167.— (1953) Proc. Soc.

lidem and Dunn, T. B. — (1956) J. nat. Cancer Inst., 16 1309
lidem AND Le\tllian, W. D.— (1950) Ibid., 10 1139

'• '■

'S. M6.-(I936) ./.

Pilgrim, H. I. — (1957) Cancer Res., 17, 405.

"^^^1? 99?lo%o?/5fi.,i[^4^‘279 9> 620.-(1959) Ibid.,

We?n AND Ht]:rsen,S.—( 1960 ) 14 907

Woolley, G. and LrrrLE, C. C.— (iks) Ibid., 5, 193 .

508

CHEMICAL INDUCTION OF MAMJIARY CANCER IN
PSEUDOPRBGNANCy

KAMAL J. RANADIVE, SAFIA A. HAiaM and KTOIUD R. KHARKAR^

From the Department of Experunental Biology, Indian Cancer Research Centre,

Parel, Brmbay 12

Received for publication May 17, I960

In our efforts to elucidate pathways of methylcholanthrene (MCA) action in
chemical induction of mammary cancer we have carried out comparative studies
on chemical carcinogenesis in several inbred strains of mice. The experimental
mice were maintained as normal or forced breeders. Analysis of this data showed
few mice that failed to conceive, but developed multiple breast tumours (Eanadive
and Hakim, 1957). This accidental observation on high tumour incidence in
sterile females attracted our attention. The sterile mice in mating cages were
perhaps in continuous state of pseudopregnancj'’. This suggestion led to further
investigation on chemical carcinogenesis in pseudopregnant mice. The present
paper is the first report on the subject.

MATERIAL AND METHOD

Three inbred strains of mice — dba(Bar), dba(-MTI) and L{P) — ^were used for
these studies. The breeding stock of the first two strains was imported from
Roscoe B. Jackson Memorial Laboratories, Bar Harbor and National Cancer
Institute, Bethesda, U.S.A., while L(P) is a line of the strain L{0) from Paris,
which was developed in our laboratories and described previously (Ranadire and
Hakim, 1958). The line L{P), resistant to spontaneous breast cancer, is found to
be particularly susceptible to JICA induced mammarj' cancer.

Small groups of available young mice of 2-3 months were used for the experi-
ment. Pseudopregnancy u'as induced in females by two different techniques ■'
(i) by ligation of Fallopian tubes in females and mating these vdth intact males ;
and (ii) by mating intact normal females udth vasectomised males. The females
from the mating cages were kept under daily observation. Frequency of forma-

EXPLANATION OF PLATE

, .1 - 11 —

Fig. 1 . — Portion of mammary ginnd whole-mount of an experirr
female, sho-n-ing mammarj- duets full of acinar-buds and two

Fig. 2. — Section of ovarj' of an experimental pseudopregnant dba(Bar)

complete leutinisation. A good number of corpora lutea are corople e j }

Fig. 3. — Portion of mammary gland whole-mount of an e.xperimenta! U^'Jvpa'^h^erpIastic
female, showing mammary duets with few acinar buds and a large loca

nodule (uD). x 19. _ , good number

Fig. 4 , — Section of ovarj' of an experimental pseudopregnant L(F) lemai s

of corpora lutea. x 24-7. . „„,,,}onr(>enant dba(Bar)

Fig. 5. — Section of portion of uterine horns of an -nj compact lamina

female, showing thin uterine horns with collapsed endometrial
propria indicating tj-pioal phase of progesterone stimulation. X 1

British Journal of CA^•CER,

Vol. XIV, Xo. 3,

CHEIMICAL INDUCTION OF MAMMARY CANCER

.ion

tion of vaginal plug was noted to ascertain the mating, and dail}' vaginal smear
record was maintained to study the nature of reproductive cycle.

The female mice of both the experimental groups received cutaneous a])j)Iica-
tion of solution of 0-25 per cent MCA m thiophene-free benzene twice a week.
The tumour incidence data on pseudopregnant mice was evaluated in comparison
vdth that of methylcholanthrene treated virgin and breeder mice of t!»e same
strains. The animals were sacrificed when they looked weak and cmacialed,
either due to a breast lesion or some skin lesions.

Gross mounts of mammary glands were prepared as described earlier (Ranadivc,
1945), and stained with haematoxjdin. Ovaries and adrenals were weighed in
normal saline, fixed in Telly’s fixative and paraffin sections were stained with
haematoxylin and eosin for routine studies. Pituitaries were fixed in Zenker-
formol and stamed with modified trichrome P.A.S. method, observations on wliich
will be reported separately.

OBSERVATIONS

(1) Psmdopregnancy

Vaginal smears . — Vaginal smear records of all experimental mice werc annlvsed
at the end of the experiment and graphs ivere prepared accordingly.

LIGATION OF FALLOPIAN TUBES

dba (Bar)

F.,nopi,„ tats L(P) and dba(Bar) „ith ligated

510

IvAMAL J. EANADIVE, SAEIA A. HAKIM AND KUMUD E. KHAEKAR

The cycle representative of both the experimental groups shoiv cliaracteri^fip
long dioestrus interrupted by a phase of short oestrus. However there is

as also m the occurrence of vaginal plugs. The Pallopian tube ligated group had
long dioestrus lasting for 10-12 days and oestrus lasting for only few horn
wlule the second group had comparatreely shorter dioestrus vdth the oestrus
lasting occasionally for 2-4 daj^s. Evidently mice from both the groups must
have gone tlirough a state of pseudopregnancy, longer in the first group than in
the second.

PITUITARY

Fio. 7, — Pathways of metliylcholanthrene action.

Uterus . — Routine liistological study of uterine horns of experimental mice
indicated specific progesterone stimulation in more than 60 per cent of the
animals. Twenty-four mice out of total 39 had tliin uterine horns. The uterine
lumen was lined by tall columnar epithelial cells with central nuclei. The
lamina propria was compact and endometrial glands were collapsed. Keratinisa-
tion of vaginal epithelium was absent. Polymorphonuclear leucocytes were
observed in the vaginal cavitj'^. The morphological and physiological findings
regarding vaginal and uterine condition were thus consistent with a state of
pseudopregnancy (Pig. 5).

(2) Breast tumour incidence

Table I gives comparative data on chemical induction of breast tumours in
vireins, breeders and pseudopregnant mice of two strains dba(Bar) and L( ).
The Table is self explanatory. The groups of treated mice of both the strains
were sacrificed between the age of 8-10 months as the ammal started becoming
emaciated and showing breast or skin lesions. Both in dba(Bar) and }
virgins, no palpable tumours were observed in the 8-10 months age group, n
breeders of both the strains, a large number of animals (60-65 per cent) developed
palpable breast tumours. The breeders developed tumours at age /-9 montns.
A noticeable difiference in the weights of the ovaries, the number of corpora Jutea,

CHEMCAL lE'DUCTIOK OF JIAMMARV CANCER

Virgins, Breeders ond
imth 20-31c(h;ilchol(in-

TyjLE 1 -Comparative 3Iammary Tumour Incidence in
l^midopregnant Mice {Fallopian Tubes Ligation) Treated
^ ihrene

Ovnrics

Strain

Tj’pe of mice
(Number)

Number of
mice with
breast
tumours

Average

age

death

(months)

9-3

Average

weight

(mg.)

3-G-bO-l

Averago
number of
cor[)orn lutea
4-7J:0-4

Dba(Bar)

. V — 7

BR.— IG

'. 10/16 ’

7*9 .

3-81i0-0!)

.0-34 4-0-28

Pseud. — 8

(G2-50;',)

8/8

(100%)

O'C

14-l±0-3

13-44-2-0

0-43±0-18

. V— 8

BR.— 17

8-0

. 1-8.54-0-25

L(P) .

'. 10/17

8-7

. 4-49±0-04

4-684-0-13

Pseud.— 1 1

(00-71%)

11/11

. 101

. 8-4±l-4

4-C0±O-2

(100%)

V = Virgin.

BR. = Breeder.

'Pi?«aiirl Pcf^ndonrotmnnt .

and granulosa cells was obseri-ed between the virgin and breeder mice treated
the carcinogen. In the strain Ij{P), the difference M'as particnlarly striking
as the virgin ovarj’' was verj’^ small and weighed hardly 1—2 mg. There were
neither well-formed corpora lutea nor granulosa cell lesions. In the breeders
many animals showed ovaries which were larger, heavier than normal with well-
developed corpora lutea and granulosa cell proliferation. Tlie e.vperimental
group of pseudopregnant mice developed palpable multiple breast tumours in
every mouse. All the eight dba(Bar) mice and eleven L(P) females developed
tumours at age 9-10 months. The ovaries, particularly of dba(Bar) presented a
characteristic picture of hea%'j’^ organs, full of large well-developed corpora lutea
(13-4: — average number). There was hardly any granulosa cell stromal element
present. The L(P) ovary also was comparatively much heavier, double the
weight of breeder ovary (8-4 mg.), with well-formed corpora lutea and granulosa
cell lesions in many. The pseudopregnant mice which mated, but did not
conceive, were virgins for all practical purposes but the difference in their ovaries
and breast tumour incidence was striking.

Table 11 gives breast tumour incidence in the pseudopregnant mice mated
with vasectomised males. The breast tumour incidence in pseudopregnant mice
of both the strains ranged between 41-45 per cent. It was higher than that in
treated virgins but comparatively much less than that in the first batch of pseudo-
pregnant mice vdth ligated Fallopian tubes.

Xf lOU U OOlU

A series of experiments carried out on five inbred strains of mice to studv the
mechanism of multistep process of chemical carcinogenesis has been described
and discussed before (Ranadive and Hakim, 1957, 1958, 1959). Fig. 7 illustrates
this data on pathways of methylcholanthrene action.

The (^remogen and/or its metabolites absorbed through the skin and /nr

fon„.tio„ be aceeleeated bylhe aoS offte SSgLw

510

KAJIAL J. EAN-aDH^E, SAEIA A. HAKIM AND KUMUD E. KHAEKAE

TJie cycle representative of both the experimental erouns show oho.. , ■
long aioootrns inlorropM by a phase

noticeaWe jhtfeienco ,n the length of the dioestra, and oestms in the’ “o
as also m the occmTence of vaginal plugs. The PaUopian tube ligated grouS
long dioestrus lasting for 10-12 days and oestrus lasting for only few hours
while the second group had comparatively shorter dioestrus -srith the oestrus
astmg occasional y for 2-4 days. Evidently mice from both the groups must
theLcond^ "^”^ ^ ^ of pseudopregnancjf, longer in the first group than in

P/TU/TARY

Fio. 7.— Pathways of methyicholantlirene action.

Uterus. — ^Routine histological study of uterine horns of experimental mice
indicated specific progesterone stimulation in more than 60 per cent of the
animals. Tweiity-four mice out of total 39 had tliin uterine horns. The uterine
lumen was lined by tall columnar epithelial cells with central nuclei. The
lamina propria was compact and endometrial glands were collapsed. Keratinisa-
tion of vaginal epithelium was absent. Polymorphonuclear leucocytes were
observed in the vaginal ca.\’ity. The morphological and physiological findings
regarding vaginal and uterine condition were thus consistent with a state of
pseudopregnancy (Fig. 5).

(2) Breast tumour incidence

Table I gives comparative data on chemical inductiori of breast tumours in
vireins, breeders and pseudopregnant mice of two strains dba{Bar) and L(P).
The Table is self explanatory. The groups of treated mice of both the strains
were sacrificed between the age of 8-10 months as the animal started becoming
emaciated and showing breast or skin lesions. Both in dba(Bar) and L(l)
virgins, no palpable tumours were observed in the 8-10 months age group, n
breeders of both the strains, a large number of animals (60-65 per cent) developed
palpable breast tumours. The breeders developed tumours at age 7-9 raontiis.
A noticeable difference in the weights of the ovaries, the number of corpora u ea,

511

CHEMICAL

IIsDUCTION OF MAMMiARV CANCER

Strain

Tj-pe of mice
(Number)

Number of
mice with
hrea.st
tumours

Average
age at
death
(months)
0-8

Average
weight
(mg.)
3-GiO- 1

Average
number of
eorjforn lutea
4-7-1-0-4

— \

Granulosa
cell l>ro.
liferation

Dba(Bar)

. Y — 'I

BR.— 16

! 10/lG

! 7-9 .

3-81i0-09

.',-.34 ±0-28

Pseud. — 8

(62 -.0%)
8/8

(100%)

9-6 .

14-li0-3

13-4i2-9

0-43-i-0-18

—

. V— 8

BR.— 17

8*0

. l-85i0-25

—

L(P) .

10/17

! 8-7

. 4-49±0-04

4 -.58x0-13

Pseud.— 11

(60-71%)

11/11

10-1

8-4iI-4

4-09±0-2

(100%)

V = Virgin.

BR. = Breeder.

Pseud. = Pseudopregnant.

and granulosa cells was observed between the virgin and breeder mice treated
tvith the carcinogen. In the strain L(P), the difference was particularly' striking
as the virgin ovarj' was very' small and weighed hardly 1-2 mg. There were
neither well-formed corpora lutea nor granulosa cell lesions. In the breeders
many animals showed ovaries which were larger, heavier than normal with well-
developed corpora lutea and granulosa cell proliferation. The experimental
group of pseudopregnant mice developed palpable multiple breast tumours in
every mouse. All the eight dba(Bar) mice and eleven L(P) females developed
tumours at age 9-10 months. The ovaries, particularly of dba(Bar) presented a
characteristic picture of hea^'y organs, full of large well-developed corpora lutea
(13-4 — average number). There was hardly' any granulosa cell stromal element
present. The L(P) ovary' also was comparatively much heavier, double the
weight of breeder ovary (8-4 mg.), with well-formed corpora lutea and granulosa
cell lesions in many. The pseudopregnant mice which mated, but did not
conceive, w'ere virgins for all practical purposes but the difference in their ovaries
and breast tumour incidence was striking.

Table II gives breast tumour incidence in the pseudopregnant mice mated
with vasectomised males. The breast tumour incidence in pseudopregnant mice
of both the strains ranged between 41—45 per cent. It was higher than that in
treated virgins but comparatively much less than that in the first batch of pseudo-
pregnant mice with ligated Fallopian tubes.

A series of experiments carried out on five inbred strains of mice to study the
mechanism of multistep process of chemical carcinogenesis has been described
and discussed before (Ranadive and Haldm, 1957, 1958, 1959). Fig. 7 illustratS

this data on pathways of methylcholanthrene action. ^ illustrates

The rarcinogen and/or its metabolites absorbed through the skin and /nr
formation nonld be .eceierated by the continnone aionTf the

512

ICAiilAL J.

RANAVWE, SAFIA a. HAKUr AND KUMVD R. KSASKAR

Table II ~Compara(ive Mammary Tumour Incidence in
piegnant Mice [Mated with Vasectomised Males) Treated with

Virgins and Pseudo-
‘20-Methijlcholanthrene

Number of

Average

Ovaries

Strain

Dhn(~MTl)

L(P) .

Tjpo of mice
(Number)

. V— 12

Pfieud. — 11

. V— 8

Pseud. — 12

mice with
brenst
tumours

5/11

(45-4%)

5/12

1 . /?n/ 1

age at
death
(months)
9-6 .

8-1

8-0 .
7-3

Average

Weight

(rog.)

4-85±0-I5

7-5±l-6

l-85±0-25

2-5±0-2

Average
number of
corpora lutea
6-33±0-I7
12-0±0-I

0-43±0-18

4-4±l-3

Clranuloja
cell pro-
liferation

V = Virgin.

Pseud. = Pseudopregnant.

Avhicli is presumed to be progesterone mimetic as also hj adequate hormonal
stimulation of the ovarj'. An indirect pathway of MCA action on the mammary
gland through stimulation of luteal function of the ovary was suggested from
obser\’'ations on ovaries of tumour mice. Significant changes were noticed in tiie
ovaries in association with development of chemically induced breast tumours in
breeders. The present experiment on pseudopregnant mice was undertaken
specially to study the action of excessiv'e luteal stimulation of pseudopregnancy
in chemical induction of mammary cancer. Although the groups of pseudo-
pregnant mice studied are rather small, they show a definite increase of tumour
incidence over that in virgins.

Andervont and Duim (1950) have succeeded in inducing tumoms in virgins of
dba(Bar) and dba(-MTI) udth MCA paintings but the incidence has been as low
as 30-32 per cent. In our experimental groups we have failed to induce mammary
tumours in 10-I2-month-oId viigtns of strains dba(E3r), dba(-MTI) and L(P).
In treated breeders of these strains the tumour incidence has been as high as
60-65 per cent (Ranadive and Hakim, 1957), perhaps because of an increase in
the luteal factor of pregnancy. In the Rallopian tube ligated pseudopregnant
mice the tumour incidence rose to 100 per cent ; all the animals developed multiple
breast tumours. This augmentation of tumour incidence may be attributed to
continuous progesterone stimulation of pseudopregnancy acting as an adequate
promoting factor.

Bonser (1954), while studying chemical induction of mammary cancer in IF
strain, intact A'irgin and ovariectomised mice, first indicated the importance of
progesterone stimulation in mammary carcinogenesis, dull (1954) soon con-
firmed, from his experiments with ovariectomised IF females, that progesterone
in combination with oestrogen acted as an essential promoting agent in carcino-
genesis by 20-MCA. He later (dull, 1957) compared this process of mammary
carcinogenesis ■with skin carcinogenesis promoted by a co-carcinogen like croton-
oil. The chemical carcinogen MCA induced the tumour cells. These latent
tumour cells grew activety only if the subsequent hormonal stimulation proved to
be adequate. Evidently in the present experiment in physiological condition oi
pseudopregnancy, continuous progesterone stimulus is adequate to stimulate
active growth in latent tumour cells of all the MCA treated mice. The findings
confirm the importance of continuous progesterone stimulation as an essential
promoting factor in mammary carcinogenesis by 20-MGA.

CHEJIICAL INDUCTION OF MAMMARY CANCER

513

The second group of psendopregnant females mated with vasectomiscd males
gave tumour incidence as low as onlj' 4 1-45 per cent. Vaginal smear datu indicated
little difference in the pattern and frequency of pscudopregnancy. J crimps the
progesterone stimulus under this experimental condition was not adequate lor
promotion of mammary carcinogenesis. Ligation of Fallopian tubes appeared to
be a better method of inducing physiologically functional pscudopregnancy.

During pseudopregnancy, the mammarj’- gland is under continuous exccs.sive
stimulation of progesterone. The gland proliferates to develop profuse acinar
budding but does not get a chance of normal lactation. According to ilfarchant’s
obsersmtions (1955) lactation has an inliibitorj' effect on the development of
induced mammai^’^ tumours in IF mice. She has latelj’’ (1958) confirmed the
protective effect of lactation in chemical carcinogenesis of mammary glands. It
is perhaps the lack of this protective action of lactation that finally results in
higher yield of chemically induced tumours under the physiological condition of
pseudopregnancy.

These observations on the importance of hormonal factor of pseudopregnancy
as a promoter and accelerator of carcinogenesis might help to explain the higher
hreast tumour incidence in nulliparous women, as well as in women of certain
communities where late marriages and prevention of lactation by not breast-
feeding the babies, is in vogue.

SU.M3LARy AND CONCLUSION

1. Pseudopregnancy has been induced in young female mice of three inbred
strains dba(Bar), dba(-i\ITI) and L(P) by two different techniques :

(а) Mating Fallopian tube-ligated females uith intact males, and

(б) mating normal intact females with vasectomised males.

Virgin, breeder and pseudopregnant mice of three strains have been given
cutaneous application of a 0-25 per cent solution of 20-methylcholanthrene in
thiophene free benzene twice a week and the incidence of breast tumours has been
studied at the age of 8-12 months.

2. None of the experimental margins, 60-65 per cent of the breeders and 100 per
cent of pseudopregnant mice of the first group, developed palpable breast tumours
Ihe tumour incidence in the second group of pseudopregnant mice was only
4y-4.‘i ner cent ^ j

3. The high incidence of chemically induced breast tumours in pseudopremiant
caSnoT^ieTi ^ promoting agLt in chemical

514

THE Dl^^ELOPJIENT OE OVARIAE^ TUMOURS IN OVAEIFS
GrRAETED FROM MICE PRETREATED WITH
DBIETHYLBENZANTHRACENE

IlvTIIBITION BY THE PRESENCE OF NoRMAL OvARIAN TiSSUE

JUNE HIARCHANT

From the Cancer Besearch Laboratories, Iledical School, Birmingham, 15
Received for publication June 25, 1960

Female mice of tlie IF strain and its Fjhybrids have been shomi to give <i
liigli jneld of granniosa-celled tumours of tJie ovary, as iveiJ as breast tumours,
after skin paintings Avitli an oily solution of 9 : lO-dimeth}^-! : 2-benzanthracene
(DMBA) (Hoiveli, Marchant and Orr, 1954). This carcinogen has since been
renamed 7, 12-dimetb3'-]benz(a)anthracene.

In a subsequent experiment, which has been preliminarily reported (Marchant,
1959a), it was shown that reciprocal bilateral ovarian grafts made between normal
mice and mice treated ndth DMBA resulted in the development of tumours in
the majority of mice with ovaries grafted from treated mice, while treated mice
bearing grafts of normal ovaries developed only breast tumours.

The present i^aper reports this experiment in more detail. It also extends it to
include the exchange of onl3’' one ovary betiveen normal mice and mice treated
with DD'IBA to see ivhether the presence of normal ovarian tissue would inhibit
the development of tumours in ovaries from mice treated with the carcinogen.

materials and method

As in the previous experiment, the mice used were 5''oung adult virgin female
Fy h3rbrids derived from C57BI mothers and IF fathers, and free from mammary'
tumour agent. Half of them received fortnight^ skin paintings of 0-5 per cent
DMBA in olive oil, about 1 mg. DMBA being given at each treatment. Six
paintings were given in all. After a further 2 or 3 weeks, both oA'aries were
dissected from the ovarian capsules of the treated mice and from an equal number
of untreated mice. The mice were diAuded into 4 groups, 2 treated and 2 un-
treated. In the experiment already reported in brief, 2 groups were described
in w'hich a bilateral reciprocal exchange of ovaries u-as made betu-een treated and
untreated mice. In the other 2 groups of the present experiment, one ovar>' of
each treated and normal mouse was replaced and the other Avas reciproealiA
exchanged. The ovaries were all grafted back inside ovarian capsules by the
technique described b3^ Jones and Krohn (1960). The mice were housed 5 to a
box and fed on rat cubes knoAvn as the Thompson diet {He3^gate and Sons) mtii
Avater ad lib. Vaginal smears Avere done on the mice at intervals. They Avere
kept until the3r died or their condition made it necessar3" to kill them. He
experiment aa^s terminated 15 months after the grafting operation. At au ops}
breast tumours were noted. Ovaries AAmre examined for tumours, then tixec ,

515

inhibition of tumoues by normal

OVARIAN

TISSUB

sectioned and stained with liaeraatoxylin and eosin.
tumourous ^vere serially sectioned and representatn o
levels examined for early tumours.

Ovaries not obviously
sections from different

RESULTS

Tlie numbers of mice in the 4 groups udneh came to
times and incidences of breast and ovarian tumours, are shoun m Table i.

Table l.-Breast and Ovarian Tumours Obtained After Unilateral or Bilateral
Exchange of Ovaries Between Carcinogen-Treated and Untreated 2hce.

Grafted

Number

Number
with breast

Hosts

ovaries

autopsied

tumours

Normal .

2 treated .

Normal .

, 1 treated .

Treated

1 normal
. 1 treated .

Treated

1 normal
. 2 normal ,

Number with ovarian tumours

Mean

, survival

Macroscopic Jlicroscopic

Cysts

(months)

14-4

14-0

3-9

6-0

1. Normal hosts bearing bilateral grafts of ovaries from DM BA-treated mice

The 18 mice in this group survived between 10‘3 and 15’3 months (mean 14'4
months) from the grafting operation.

None of the mice developed breast tumours. Near the time of deatli, several
of them had fairly constant oestrus vaginal smears.

Ovarian grafts . — In 14 of the 18 mice solid ovarian tumours were found in one
of the grafted ovaries. Thirteen were macroscopic, most of them being I cm. or
more in diameter. Six of these 14 mice had large cysts filled with clear fluid in
the contralateral ovarian grafts. Of the 4 remaining mice, one had bilateral
cysts and the other had only atrophied remnants of ovarian tissue.

Histologicallj', 1 of the 14 tumours was a haemangioma. Five were tjqiical
granulosa-celled tumours. Five others were granulosa-celled tumours whose
cells showed varjdng degrees of luteinisation and the remaining 3 were almost
completely luteinised.

Oestrus or suboestrus vaginal smears were shoum by the majorit}' of the mice
with solid ovarian tumours, but not by those uithout.

2. Normal hosts bearing grafts of one normal ovary and one ovary from a DMBA-
treated mouse

The 1 6 mice in this group survived between 14 and 15 months fi-om the graftinff
operation (mean 14-9) months. The experiment was terminated at 15 montlis to
make tins group comparable with the previous group

developed breast tumours. Vaginal smears done near the
<o\ ^^\^^™ble, but few showed oestrus activity at that time.

(a) Grafts of ovaries from DMBA-treated mice — In 4 of the Ifi mino f

JUNE MAECHANT

516

celled tumour, as was 1 of the other nodules. The remaining 2 very small nodules

of granulosa-celled tumour were not luteinised. ^ imoaules

A further 7 grafts of ovaries from treated mice had developed into cysts filled
vith clear fluid, varying in size, but of the order of I cm. diameter. Another one
had become a fibrinous clot. In the walls of the cj^sts, the onfy recognisable ova-
rian tissues remaining were heavily pigmented lutein cells and, occasionally
Jiya inised fragments of corpora lutea. Similar tissue was found in the remnants
01 the other 4 ovaries grafted from DMBA-miee.

(b) (?n7//a of normal ovaries replaced in their hosts.—None of the normal
ovaries replaced in their normal hosts contained tumour nodules.

In 9 of the 16 animals, cysts filled with clear fluid arose, which were often 1
cm. or more in diameter. In their walls, remnants of pigmented or hj'alinised
lutein tissue were found. Tour of these cj^sts were in mice which also had cysts
in their ovaries grafted from DMB-treated mice. The remaining 7 grafts were
atrophied and di0’usel3’’ luteinised and 4 of them contained remnants of hyalinised
corpora lutea. In 3 of the grafts a ver}'' few small follicles were found.

3. DM BA-treated hosts bearing grafts of one normal ovary and one ovary from a
DM BA-ireated mouse

The 13 mice in this group survived between 1 and 5 months (mean 3-9 months)
from the grafting operation. The short survival time was due to the appearance
and rapid growth of breast tumours in all 13 mice. Nine of them developed
multiple breast tumours, 2 having as many as 5 tumours.

Histological examination of the tumours showed that the}' were adenocarcino-
mas, frequentl}'- of the papillary cystic tj'pe, irith abundant fibroblastic stroma
and slight secretion. Squamous metaplasia rvas not frequently seen and sebaceous
metaplasia •was only seen in one tumour.

Vaginal smears were again very variable, but vith little evidence of oestrus
activit3^

(a) Graffs of ovaries from DMBA-ireaied mice replaced in their hosts. — In 1 of
the 13 DMBA -treated mice a nodule of granulosa-celled tumour was found in the
host’s own replaced ovai^'’. Clear fluid-filled C 3 ’sts 0-5 to I'O cm. diameter with
pigmented cells in the c 3 ’-st v'alls were found in 6 others. The remaining 6 grafts
were ver 3 ^ atrophied and were composed of a few pigmented cells.

(b) Grafts of ovaries from normal mice.—'No tumours developed in grafts of
ovaries from normal mice implanted in DMBA-treated hosts. In 5 animals the 3 ^
developed into cysts filled Avith clear fluid. In the walls of the cysts normal
ovarian tissue, composed mainty of follicles and corpora lutea, v'as found. m
remaining 8 grafts of normal ovaries were similarly composed and they showed

slight at^oph 3 ^

4. DMBA-treated hosts bearing bilateral grafts of ovaries from normal mice

The 17 mice in tliis group survived 2-5 to 11 months (mean 6 months) from the
grafting operation. All except the mouse dying after 2-5 months developed
breast tumours, 7 of them having multiple tumours.

HistoloaicaUy the breast tumours were adenocarcinomas, usually with a buna
ant fibroblastic stroma and slight secretion. Foci of squamous metaplasia i
occasionally seen.

inhibition of tumours by normau ovariam tissuu

nl'

Vaginal smears showed infrequent periods of oestrus. No Uunours were

i of the 17 mice imilateral eyets Mere fouml. They Mere
O S^ri o cm in diameter and iilled rvith clear fluid. Follicles acre ,)rcsont in
the evsts walls. The other ovarian grafts were somewhat atrophied but noi m
ovariln structures were present in them, namely follicles, corpora liitea, pigmentc
cells and, in some of the older grafts, hyaline corpora lutca cliaractcnstic of

C57B1/IF hybrids.

DISCUSSION

Of the 18 normal mice of group 1 bearing bilateral ovarian grafts from mice
treated vdth DiilBA for 3 months, 14 (78 per cent) developed ovarian tumours,
13 being macroscopic. When treated ovaries were transplanted, a small pro-
portion of them probably contained early tumour nodules. A previous experiment.
(Marchant, 19596) in which DMBA-treated mice were killed at monthly intervals
showed the earliest detectable tumour nodule in 1 of 10 mice killed after 3 months,
after which there was a roughty linear increase in the proportion of mice ivith
tumours as time increased. It is certain that many of tlie 14 tumours in tlie
present experiment must have arisen from ovaries in which no detectable tumour
nodules were present at the time of grafting. The environment of tlie untreated
hosts whose ovm ovaries had been removed allowed the development of such
nodules and their growth to a large size.

The untreated mice of group 2 bearing grafts of one normal ovary and one
from a DiEBA-treated animal survived a similar length of time to those of groiqi
1. However, no macroscopic ovarian tumours developed and microscopic tumour
nodules were found in only 4 of the 16 animals (25 per cent) in the ovaries grafted
from treated mice. It is considered likely that these tumour nodules were all
present at the time of grafting, since a small proportion of animals previously
killed after 3 months DiNIBA treatment had tumours (Jlarchant, 19596). How-
ever, the fact that no macroscopic tumoiu-s developed reveals tlie inhibitor^"
influence of the normal ovaries grafted on the contralateral side.

Group 3 also shows this inhibitory effect. In the 13 DjMBA treated animals
grafted vdth one normal ovary and one from a treated animal, onty 1 microscopic
tumour nodule was found in an ovary from a treated animal. The survival of
this group of animals was 3-9 months from grafting and this should have heen
ample time to jdeld a high proportion of tumours from the pretreated grafts if
no mhibitorji- factor had been present. The single microscopic tumour found had

gnJting ^ treatment prior to the time of

yio inWbition by normal ovarian tissue of tumour development in ovaries
grafted from mice pretreated tvith DJ'IBA falls into line tvith theMnbS rf
ovarian tumour development by other methods. For instance after PriftinD- nn
ovarjf into the spleen of castrated rat tumours uill appear in th^ lafts Jut these
arc prevented if one ovary is left intact (Biskind and Biskfrd i f

the induction of ovarian tumours in minp nffoT' 1 Similarlj ,

found to inhibit ovarS;;- ium^urifeSnLnS;,“"^^^^^^^^

518

JUNE MAECHANT

Gardner, 1949) and after X-rays (Gardner, 1950). However, when stiJboestroI
dipropionate was administered simultaneously wth DMBA, it failed to inliibit
the development of ovarian tumours (Marchant, 1957). In this case the hormone
Avas administered aiong ivith the carcinogen by skin paintings of a mixture of the
substances in olive oil. This method of administration may have been
ineffective because of different rates of assimilation of carcinogen and hormone
tlie hormone probably being metabolised aivay more quickly than the carcinogen
on account of their differing solubilities.

SUMMAKY

First generation hybrid mice from C^Bl mothers and IF fathers ivere given
pretreatment of 6 fortnightty skin paintings of olive oil, each containing 1 mg.
of 9 : lO-dimethjd-l : 2-benzanthracene (DMBA). Both of their ovaries ivere then
reciprocally exclianged Avith those of similar untreated mice, or one ovary only
Aims exchanged and the other reimplanted. The grafts Avere made into the
OA'arian capsules.

Of the IS untreated mice Avdth bilateral grafts of pretreated ovaries, 14 dei^elo-
ped OA'arian tumours, 13 being macroscopic. In tlie 16 untreated mice Aiith
unilateral grafts of pretreated OAmries and contralateral normal oAmries, 4 micro-
scopic tumours AA^ere found in the grafts of pretreated OAmries. In 13 DMBA-
treated mice Avith similar unilateral grafts, only 1 microscopic ovarian tumour
Avas found in a pretreated OAmry. The 17 DMBA-treated mice AAuth bilateral
normal ovaries did not develop any ovarian tumours.

It is considered that the presence of the grafts of normal ovaries inhibited the
deAmlopment of ovarian tumours in the raice-Aidth unilateral grafts from mice
pretreated Aidth DJIBA. The small number of microscopic tumours found were
jirobably present at the time of OA-arian grafting.

I am grateful to the Birmingham Branch of the British Empire Cancer Cam-
paign for support of this AA^ork.

REFERENCES

Biskind, G. R. and Biskind, M. S.— (1948) Science, 108, 137.

Gardner, W. U. — (1950) Proc. Soc. exp. Biol. N.Y., 75, 434.

Hoaa’^ell, J. S., Marchant, J. and Obr, J. W. — (1954) Bnt. J. Cancer, 8, 635.
Jones, E. C. and Krohn, P. L.— (1960) J. Endocrin., 20, 135.

ICaplan, H. S.— (1950) J. nat. Cancer Inst., 11, 125.

Li, M. H. and Gardner, W. U.— (1949) Cancer Res., 9, 35.
lidem and Kaplan, H. S. — (1947) J. nat. Cancer In^., 8, 91.

Lick, L., Kieschbaum, A. and jVIixeb, H.— (1949) Cancer Res., 9, 53^.
Marchant, J. — (1957) Brit. J. Cancer, 11, 452.

Idem.—{lQ5Qa) Acta Vn. hit. Cancr, 15, 196.

Idem.—(lQ59h) Brit. J. Cancer, 13, 652.

Tm tojwxoPMENT of ovaeian tumours in ovaries
the DETOMUmAi PEBTEEATED WITH

DBIETHYLBENZANTHRACENE

The Effects of the Geafting Operatiox Itsei.f akd the Effects
OF Grafting Ovaries from Mice at Earia' Stages ix Pretreaimfv*

AVITH THE CaRCINOGEX

JUNE MARCHANT

From the Cancer Research Laboratories, Medical School, Birmingham, l.o
Received for publication June 25, 19G0

Skes painting of female mice of the IE strain and its Ep hybrids with an oily
solution of 9 •. 10-dimethyl-l 2-benzantliracene, now renamed 7, 12-dimethyl-
benz(a)anthracene, induced breast tumours and granulosa-celled tumours of tlic
ovary in a high proportion of animals (Howell, Marchant and Orr, 1 954-).

In a subsequent experiment (Marchant, 1959a, 1960) oAmrian tumours aro.se
in 14 out of 18 normal mice Avhose own o\mries were removed and replaced with
those of mice which had been pretreated Avith the carcinogen over a period of 90
days. Large cysts filled with clear fluid were also found in 7 of these animals.
Another experiment (Marchant, 19596) had shown significant changes in ovaries
examined after 90 days of pretreatment Avith the carcinogen. The5’’ were atro-
phied, with almost complete destruction of follicles and diifuse luteinisation,
and early tumour nodules had just begun to make their appearance.

The present experiments AA'ere designed to discoA'er Avhether ovaries grafted
in much earlier stages of pretreatment AAith the carcinogen would deAmlop ovarian
tumours and cysts, and to see whether the grafting operation itself was effective
in giA’ing rise to tumours or cysts.

jlaterials axd jiethods

All the mice used in this investigation were young adult virgin females lacking
the mammary tumour agent. They were Ep hybrids derived from CspBl mothers
^d IE fathers. They were housed 5 in a box and fed on rat cubes, known as the
Thompson diet, -with water ad lib.

The donor mice were given skin paintings of a 0-5 per cent solution of
dmirthylbenzanthracene (DJIBA) in olive oil once a fortnight, commencing Avhen
the} u ere .. to 3 months old. About 0-2 ml. (containing 1 mg. DIMBA) ivas given
a each treatment, distributed over dorsal and Amntral surfaces of the bodv

carcinogen treatment, the

bihterallv^to ^ carcinogen-treated mice and grafted

^ --s cm

grafts of ovaries from untreated donors ‘ ^ ^ ^ ^ ^ received

520

JUNK MABOHANT

the OYarian grafts were examined for tumour and histological
lor microscopical examination.

material was taken

KESULTS

The results of bilateral grafting of ovariectomised
DMBA-treated donors are summarised in Table I.

mice irith ovaries from

Table l.~Besidts of Bilateral Grafting of Ovaries from Mice Treated Fortniqhthi
with DmetJnjlbenzanthracene {DM BA) for Different Periods to Normal Hosts
Whose Own Ovaries Were Removed.

Grafted ovaries
(days since 1st DJIBA)

Untreated

Number
autopsied

8
G

0 ,

10 ; 0

jMicroscopic Cysts

2 ’s

1* 3

1 * 2

0 4

1 C

0 S

Number with ovarian tumours

Macroscopic
1

8 *

* Bilateral tumours.

Mean

survival

(months)

14' 1
IT'8
17’3
18.7
15-8
lO'O

Mice with ovaries grafted 10 days after first D^IBA-ireatment

Tour mice in this group were autopsied in a mean time of 14-1 months (range
13|- to 14^). In one of these animals a pseudofoUicular granulosa-celled tumour
with a mean diameter of about 2-0 cm. was found in one ovary. In 2 others,
nodules of undifferentiated granulosa-celled tumour tvere found in the walls of
large, fluid-filled cysts. In 1 of these mice the contralateral ovary was also cj^stic ;
in the other it was verj'- atrophied and yellow and composed of a few large pig-
mented cells, as were most non-tumourous ovaries in the experiment.

In the fourth mouse a large cyst filled noth clear fluid was found in 1 ovarj% but
there was no tumour tissue. Vaginal smears of these mice all showed evidence
of oestrus activity near the time of death.

Mice with ovaries grafted 20 days after first DMBA-treatment

Seven mice in this group were autopsied after a mean time of 17-8 months
(range 13-3 to 21 months). All of the mice in the group developed ovarian tum-
ours varjdng in size from 0-4 cm. diameter to 1 4 vith 3 lobes, each lobe being over
1 cm. diameter. Two mice had bilateral tumours. Three mice had cysts filled
with clear fluid in the contralateral oymrj^ while in the remainder the second ovarj'

was very atrophied and yellow. i • i ■ i

The tumours were granulosa-celled, but showed a variety of histological
structure. One small tumour was relatively undifferentiated. The large, three
lobed one was partly undifferentiated, partly pseudofoUicular and partly cribri-
form with some luteinisation of the tumour ceUs. Most of the remainder showec
a tendency to a tubular arrangement of ceUs with varying degrees of luteinisation
of them. Four of the 7 mice showed evidence of oestrus or suboestrus activity
near the time of death.

DEVELOPXtENT OF TUMOURS IX (?RAT' I EO

OVARIES

521

Mice with ovaries grafted 30 dai/s after first DMJ3A treatment

Eitrht mice came to autopsy in a mean time of 17-3 months
•70-8) ° All developed ovarian tumours vnrj-ing in size from O-l cm. to w ell o\ it
I cm', diameter. One mouse also had a tiny tumour m the contralateral ovary.
Two others had cysts about 1 cm. diameter m t he second ovarv'. I n o
were solid, undifferentiated granulo.sa-celted tumours, 3 were pseudofolheiilat
and 2 were ver\" heavily luteiniscd, the other 2 having the tubular niTangemenl
of cells. Eon-tumourous ovaries were atrophied and pigmented. Nngiiial
smears showed some evidence of oestrus activity, except in the mice with the
hear-ily luteinised tumours.

Mice u'ith ovaries grafted 40 days after first DM BA treatment

The 6 mice in this gi'oup survived a mean time of 1 8-7 months (range 14 -a
to 20-8). Three of them developed macroscopic tumours in 1 ovary, 2 having
large C3'sts in the contralateral ovart'. A fourth mouse had a large cyst in I
ovary ; the fifth had 2 cj'stic ovaries about 0-3 cm. diameter. In the si.xth, only
atrophied pigmented ovarian remnants were found.

The large tumours were granulosa-cellcd with luteinisation in the cells of 2
of them, the third being verj' haemorrhagic. Oestrus activity was detected in
2 of the mice vith tumours, but not in the other 4.

illfce with ovaries grafted 60 days after DMBA treatment

Nine mice in this group were autopsied in a mean time of Iti-S months (range
12-5 to 19-o). All had ovarian tumours. Two had bilateral macroscopic tiiinoiirs
and in 1 of these mice 1 of the tumours was attached to a large cj'st filled with
clear fluid. Tavo other mice had similar large tumours attached to large evsts
in 1 ovary only, the other being atrophied. Four had macroscojiic tumours in
1 ovary only and 2 of these had cj'sts in the other. The last mouse had large
bilateral cysts and the wall of 1 contained tissue in which a small tumour was found
microscopicall}’^.

Tavo of the tumours in these animals AA'ere rather undifferentiated granuiosa-
celled tumours, vdth some degree of luteinisation of cells. With the exception of
1, the remainder had the tubular structure already described, Avith variable
luteimsation. The exception Avas an oyary about 0-4 cm. diameter composed of
corpora albicantia ”. Although this ovarj- Avas undoubtedlv enlarf'cd it Avas
not considered neoplastic and has been excluded from the tumours in the table
of results A simdar coition is frequently seen in the ovaries of C57BI /IF mice

Mice with bilateral grafts of ovaries from untreated mice

ours. Five mice haiTbilateraf macrosconie I developed ovarian tum-

imilateral ones. The remaim'ncr r. • fluid-filled cysts and 3 more had

of diffusely luteinised tissue, often herTily pimSteil"' S'’,™”!’ composed
»cre found ,n 3 of them. A «ele teas iJ/Tn 1

522

JUNE MAECHANT

Without cystic ovaries. One mouse mth bilateral cysts developed a' mammar.-
adenocarcmoma after 18 months, the only spontaneous breast tumour found ?o
far m mice of the C57B1/IF constitution.

The majority of mice in this group did not show any evidence of oestrus
activity, as judged by vaginal smears near the time of death.

niscnssioN

The experiments reported above show that a liigh proportion of tumours
can be obtained from ovaries grafted at much earlier stages in pretreatment of
mice 'ivith DMBA than the 90 days in the original grafting experiment. Those
grafted to normal mice only 10 days after a single skin painting of 1 mg. of D5IBA
in oh've oil were able to proceed to subsequent neoplasia in the majority of animals.

In an investigation of the ovarian clianges occurring during treatment of mice
with this carcinogen, it was not possible to detect an^dliing other than precocious
ageing of the ovaries in the earlj’^ stages of treatment (Marchant, 1959b). There
was atrophy of tissue due to rapid loss of follicles, followed by reduction in corpora
lutea, with their fusion and disappearance or hyahnisation, and the appearance of
pigment. The earliest tumour nodule was detected at the stage in DilBA treat-
ment when follicles and ooc 3 d;es had almost entirely disappeared and the tumours
steadily increased in numbers from then on.

In the experiment reported above, in which normal ovaries were grafted to
normal mice whose own ovaries had been removed, histological examination
revealed only 2 follicles in 20 ovaries. In most cases the ageing of the ovarian
tissue had proceeded to a stage comparable with that reached after several montlis
of DMBA treatment, when a considerable number of tumours or tumour nodules
would be found in DMBA -treated mice, but no neoplastic nodules were detected
in these grafts of normal ovaries. This suggests that the induction of ovarian
tumours in mice treated with DMBA is primarily a result of some phenomonon
additional to, or quite apart from, the ageing effects -with their consequent changes
in hormonal balance which occur after treatment with this carcinogen.

The grafting of normal ovaries did give rise to the development of large fluid-
filled cysts in the majority of animals. Further evidence for this was found in
another experiment in which normal ovaries were grafted to nuce treated with
DMBA (Marchant, 1960). Again no trace of solid tumour nodules was found.
The appearance of these cysts in a series of ovaries which did not develop any
granulosa-celled tumours would appear to exclude them from the same categoyv
of growth. They were extremely thin-w'alled and transparent and, when pierced,
they collapsed leaving an amount of tissue no greater than would be found m
atrophied non-c 3 >^stic ovaries. Jones and Krohn (1960) found small flmd-mle
cysts in orthotopie ovarian autografts after 25 da 3 ^s or less. They considered them
to have arisen from proliferating cells derived from the germinal epithehum.

SOTIIUAKY

First generation hybrid mice from Cj^Bl mothers and IF fathers were gwen
fortnishtlv skin paintings of olive oil, each contaimng 1 mg. of 9 : lO-dimeth}!
fAwLttocLe (MIBA). At tatervak of 10, 20 30. 40 onl ®
the. begiiiiiing of treatment, both ovariea were removed from a group ol mice ana

DEVELOPMENT OP TUMOimS IN CHAFTEl) OVAKIES

transplanted to the ovarian capsules of untreated hybrids whoso own ovaries had
been removed. Another group of untreated hybrids received grafts of ovaries
from untreated mice.

A high proportion of mice bearing bilateral grafts of ovaries from DMMA-
treated mice developed ovarian tumouns, most of them being macroscopic. 'I'he
proportion of mice bearing tumours in each group <lid not. vary great ly wit h t lie dura -
tion of pretreatment of the ovarian grafts. No ovarian tumours develojicd in
mice grafted with normal ovaries. Large fluid-filled cysts developed in several
mice in each group, including those grafted with normal ovaries.

This work was supported by the Birmingham Branch of the British hhuiiire
Cancer Campaign.

REFERENCES

HovmLL, J. S., JIabchant, J. ano Oru, J. W.— (fO.ot) Brit. J. Cnnrrr. 8.
Jones, E. C. and Keohn, P. L. — (1900) J. Endocrin., 20, lll.'i.
jMaechant, j.— ( 1959a) Ada. Un. int. Cancr., 15, 196.

Zdem.— (1959b) Brit. J. Cancer, 13, 652.

Idem. — (I960) Ibid., 14, 514.

524

QUANTITATRT5 STUDY OF
WITH TISSUE GROWTH.
VARIOUS PHYSIOLOGICAL

A SERUM PROTEIN ASSOCIATED
LEVELS FOUND IN RATS UNDER
CONDITIONS

D. A. DARCY

Frojn the Chester Beatty Research histitide. Institute of Cancer Research,
Royal Cancer Hospital, Fulham Road, London, R.JF.3

Received for publication July ID, JBSO

Ix a previous paper (Darcj’-, 1957) a study was presented of a rat serum protein
wiiose titre was very greatly increased in the blood serum of tumour-bearing rats
and to a lesser extent in the serum of rats which were undergoing regeneration,
wound-healing, pregnancj'', or in verjr young rats. It was suggested as a working
hypothesis that tliis substance was directly associated vith cell division. It was
soluble in sulphosah'cjmlic acid and moved electrophoretically with the a-globulins.
This work was confirmed and extended by Campbell, Kernot and Roitt (1959) in
the rat, and supported by Glenn, King and Marable (1959) who worked with
human serum.

The present study is a quantitative confirmation and extension of the earlier
semi-quantitative results. It is based on an immunological method developed
for the measurement of specific proteins in serum (Darcy, 1960a). It sets out
to provide a base-line of values for this particular protein in the serum of untreated
healthj'’ adult rats, for pregnant rats, and for very young rats. In the following
paper (Darcy, 19606), values will be given for tumour-bearing rats ; in subsequent
papers the behaviour of the protein in rats undergoing regeneration and wound-
healing, and its physicochemical properties wiU be described. Its actual identity
has not jmt been estabhshed but of the knowm serum a-globulins it will be shown
to resemble most closely the protein called “ fetuin ” by Pedersen (1944a, 1947)
ivho found it in foetal calf serum, w'here it forms 45 per cent of the serum protein.
Fisher, Puck and Sato (1958) later showed that fetuin was present, although in
much smaller amounts, in human adult serum. Meyers and Deutsch (1955),
howwer, found that fetuin contained at least six components by immunologkal
analysis, ■whereas the present protein appears to have only one (Darcy, 1957).

In addition to the work on the specific protein, some findings are presented
about the total serum proteins of foetal and jmung rats and their mothers, about
which little has been hitherto published.

MATERIALS AND METHODS

Animak.— Healthy albino rats were used except where otherwise stated.
They %vere from a colony originally derived from the Wistar strain , tlie co on\
had been subjected to a degree of inbreeding in the past but -n'ere current j pro
pa gated by cousin mating. They were remarkable for their large size ^d ro i
health. They wUl be referred to as the C.B. (Chester Beatty) rats. They we c
fed once per daj^, between approximately 9.30 a.m. and 10.30 a.m, wee ug .

SEBUM PROTEIN ASSOCIATEII ^Y1T1I TISSUE UROWTll f-i-'

inbred strains n-erc also tested, the Wistar, the August and the Marshall, as rv.dl

as an FI cross between the last two. • i <

£/eedi«o.— Animals were bled from the heart under et her anae.s hesia l)etn( en
10 am and noon. The technique devised for bleeding foetal and newborn rats
was 'a modification of the capillary tube method. The animals were remov.a
from their mothers, wiped clean, the heart exposed and punctured so hat. it.
bled into the thoracic cavity ; from there it wa.s collected by iqijilymg 'P
of a capillarj^ tube of large size (2 mm. internal diameter, 0 c.m. J '>'>

(ky end of the tube was sealed in a flame giving a “ test.-tubc in which the Idood
clotted and was centrifuged. The tube was then cut just above the clot and the
serum fed into the tip of a graduated 0-1 ml. piiictte.

Measxiremenf of senim protein.— Total scrum proteins were determined by the
copper-sulphate method of Phillips, van Slyke, Hamilton, Dole, I'jiner.son and
Archibald (1950). It was first compared with the Kjeldahl method and found
to be accurate for the rat sera. The Kjeldahl method was used for 17 day foetal
serum because of its low protein content. The s])ccific protein was deteriniiied
by the method referred to above (Darcj% inOOu), which is a sinqilc c|iianlitativ(‘
application of the gel diffusion technique of Ouchterlony ( 1 94S). An average error
of about ± 15 per cent (95 per cent confidence limits) was obtained for routine
titration and this was improved in the later part of the work to about — 7 per
cent. The titres are expressed here in arbitran,’ units. A unit is defined ns the
amount of the protein in one milliliter of a 1 per cent solution of the acetone-
precipitated proteins of a particular sulphosalicylic acid extract of serum from
Walker tumour-bearing male rats.

Two preparations of the protein were used for the production of antisera,
the starting material being serum of rats bearing the Walker tumour. One was a
sulphosalicylic acid extract of the cancer serum ; it contained the ])rotoin along
noth several others. The second was purified preparation obtained by the electro
phoretic method of Laurence (1956) at pHo, after preliminarj' ammonium suljihate
precipitation to remove albumin. The jdeld by this method was small and the
reproducibility poor. Nevertheless the sample so obtained gave a virtuallv
monospecific antiserum with which most titrations were done. The antisera
prepared unth the sulphosalicylic acid— extracted protein showed several pre-
cipitate bands in the Ouchterlony plate. The specific band could be easiK'
recognised because it was the one nearest the antiserum depot when the nroteiii
extract or serum of -Walker tumour bearing rats was run against these antisera

Normal male rat serum

rate at 0 day LI a fe,v LZ dd ft fee f SL"' liT ''“'t ''
vidiial rats except those of the 17 dav ^ from indi-

pool of the sera of aU the males of one^litter The^r^^n each serum represents the
p™tci„ for the 17 day fc.tal sera a^d fc hatft h?"

about -^2 f per cent, foetal sera was only

526

D. A. DABCY

level of about 0-23 between 8 and 10 weeks after birth. The specific urotpin
therefore U times as high in the blood serum of the 1 week old rat as in the vouni
adult ; its proportion to the other serum proteins is over 7 times higher at i
week than at 10 weeks. °

LE I. — Level of the Specific Protein and Total Protein

in the Serum of 2Iat(
deviations and Number

C.B. Rats at Various Ages. The
of Sera (in Parentheses) are Given

Means, Standard 1

Ago of rats

Specific protein

Total serum protein

Specific protein

(units /ml.)

(g./lOO ml.)

-fr-: 1 — — X 100
total protein

— 5 days

. 0-2G3±0-045 (4)

2-43±0-20(3)

10-S

— I day

. 0-725±0-220 (20)

2-52±0-18 (18)

28-8

0 day

. 0-80G±0-129 (22)

2-04±0-20 (25)

30-5

1 week

. 0-8GG±0-170 (10)

2-93±0-3G (10)

29-5

2 weeks

. 0-598±0-291 (10)

3-91±0-45 (10)

15-3

3 weeks

. 0-522±0-186 (10)

4-58±0-44 (10)

11-4

4 weeks

. 0-320±0-137 (10)

4-88±0-38 (10)

G-G

6 weeks

. 0-295±0-0G7 (10)

5-31±0-32 (10)

5-6

8 weeks

. 0-245±0-0G9 (23)

o-89±0-28 (23)

4-2

10 weeks

. 0-231 ±0-033 (12)

5-82±0-17 (12)

4-0

12 weelcs

. 0-269±0 040 (12)

5-57±0-26 (12)

4-8

IG weeks

. 0-231±0-040 (12)

6-25±0-25 (12)

3-7

It is of interest that the specific protein was at a low level in the 17 day foetus,
but had increased dramatically in the 22 day old foetus (—1 day). The low level
in the 17 day foetus was not merely an absolute one but w’as also relative to the
other serum proteins at the time. In this respect the specific protein differs from
fetuin in calf serum, which reaches its highest level in foetal serum (Pedersen,
19446). The two proteins are alike, however, in falling sharply in the weeks after
birth.

It is reported below that fasting for about 20 hours may produce an increase
in titre of the specific protein. It is possible that the irregularities in the titre
curve (e.g. at 12 weeks) are the result of differences in the state of feeding of the
rats when bled : some of the rats may have broken their fast sufficientlj’’ to lower
their titre.

An observation made continually tlmoughout this investigation was that the
titres of specific protein for any ghmn age group of rats, when plotted as a fre-
quency distribution, rarety gave a symmetrical curve of error, but rather a skew
curve with its long arm towards the higher values. The most probable explanation
is that individuals with these exceptionally high titres were carrying some hidden
infection or other abnormality which caused the increase.

Comparison of various rat strains

Table II shows the level of the specific protein, together with the total serum
protein and mean rat weight, for rats 8 weeks of age. Tlnee inbred
Wistar, the August and the Marshall strains were tested, and an Fl hj^brid e ^veen

two of these. . . r if ilw

First a comparison was made between the male and the virgin lernaie °
C.B. stock rat at 8 weeks of age. The female shows a value for the specific pro ei
83 per cent liigher than the male, and the difference, by the <-test is hig i J i
cant (P <0-001). Lest this should have been due to chance selection, the poo

SERUM PROTEIN' ASSOCIATED WITH TISSUE OROWTH

[in Parentheses) Are Shown

Rats

C.B. <S
C.B. ?

MTstar ^
August S
Marshall ^

Specific protein
(units/ml.)
0-245±0-0G!) (23)
0.449i0-077 (13)
0-251i0-055 (9)
0-25o±0-119 (23)
0-23l±0-025 (9)

August-Marshall 0- 323^0 '137 (10)
F1<J

Total scrum protein Spec[fii^Iirr)teJ|i
(p./inn ml.) Total iirolein

.o-89-i-0-2S(23) .

G-21i0-18 (13) -

100

.5 ir>±0-30 (0)
3-21±0-21 (21)
5-41i0-fi7 (0)
5-97i0-2(i (10)

4-90

4-33

4-27

r)-4.7

Mean rat
weight
((-’•)
233
21 S
122
117
111
121

serum of 48 C.B. females Gj tveeks old was titrated and fonnd to liave n vnlut' of
0-48 ■ this agrees well tnth the value in the table (0-440) sinee the younger fcniale.s
would be expected to show a slightly higher value. Later tlirce otlier pooled
sera from C.B. female rats averaging about 8 weeks of ago were fotind lo liave liie
values 0-450, 0-442 and 0-404.

The value for the specific protein of the male C.B. rats does not. dmei
significantly from that of any of the other strains. With greater nunibei.s and
ad lib feeding a significant difference might be establi.shcd, c.g. between tlio C.B.
and the August strain. The failure to obtain such a difference is due to t he vari-
ability of the titre from rat to rat within a strain. Nevertheless the mean value
does not differ greatly from strain to strain -, none of the 3 inbred strains diffcr.s
from the C.B. by more than 9 per cent. This is noteivortliy in view of (he fact,
that C.B. rats weigh about twice as much as the other three-strains at this age.

The variability is considerable and is in general no less in tlic strictly inbred
lines or the Bl than in the C.B. stock. The high mean r-alue for the Fl (August -
Marshall) just failed to reach a significant difference at the 5 per cent level from
that of either parent strains. It was noted that the values for the ten Fl fell into
two sharply defined groups, one centering around 0-25 and the other around 0-50.
Since these animals must be presumed to be alike genetically, it can only be as-
sumed that those vith the liigh values were suffering from some undctectcii disease
or other stress. In this connection it is of interest that one of the Wistnr rats
which had to be rejected before the experiment because it had a wound on its
muzzle and could not therefore be regarded as normal, gave a value for the specific
protein of 0-51, i.e. twice the mean value for the other animals and significanth'
different from it. The total serum proteins for this rat was 4-86 g. per 100 ml',
i.e. below the average. It should also be pointed out that the ratio of the siiccific
protein to total serum protein is fairly constant from strain to strain e.xcerit for
the high value in the August-Marshall Fl. The C.B. females, of course stand

Qrpfl,rt. *

, August rats had been bled in three groups : one group of 4 bled in October
vake^of 0-20™^fr December 1959 gave a mean

oS" tL tnceS e i„ 1*3/ value of

value, and 1 1 “ ra"uLS '>5'

characteristic for a strain but that hi Jf ^ ^

as a result of some^freaTemstes.^^^.''^*’'"" occasionally occur probably

528

D. A. DARCY

Effect of fasting

• .u mfe.— The effect of fasting on the level of the specific protein is slioirn

m the fblloTOng experiment. A batch of August rats 12 weeks of age iras divided
by random selection into tivo groups, one of 7 and one of 6 rats. They were all
bled at the same session and under the usual conditions except that the groun'of
6 had been fasted for 22 hours, the food which remained in their cages havint'
been removed. The other rats had enough food to last most of the day The
results are shown in Table III. It vdll be seen that the fasted rats have a level of
the specific protein which is about 35 per cent higher than the controls. The
difference is significant at P = 0-02. The differences in total serum protein and
body M'eight were not significant.

Table III. — The Effect of Fasting Upon the Level of
Specific Protein in the Serum of 12 Weeh Old 3Iale August Pais

Specific protein

Total serum protein

Bat weight

Number

(units/ml.)

(g./lOO ml.)

(g-)

Mean S.D.

rats

Non-fasted

0-187±0-035

6-52±0-39

lS5-9±8-l

Fasted

0-253±0-049

6-00±0-lG

147-2±1M

P .

0-02

0-2-01

>0-1

C.B. rats . — The experiment was repeated for the C.B. rats but in a somewhat
different form. Twenty -four C.B. males, 8 weeks of age, were separated by random
selection into two boxes. Since the normal method of feeding of these animals
means that they are fasting for about 20 hours by the time the}’^ receive their
single allotment of food in the morning, the rats in one box were simply allowed
the normal quota of food ivhile the others ivere given food ad lib. All animals were
bled the next morning without further feeding ; there was still food remaining
in the box of the ad lib fed rats and also in their stomachs, but none in the stomachs
of the fasted rats.

The results are shovTi in Table IV. The ad lib fed rats were significant!}''
heavner than the fasted ones ; their serum protein reached a higher (but not
statistically significant) total than the fasted ones — an interesting difference from
the August rats. The specific protein was 48 per cent higher in the fasted rats,
yet this difference was not significant (P = OT). However, there was one extrem-
ely high value among the fasted rats, namely 0-945, which can only be accounted
for on the basis of some undetected disease or other abnormality. If this value
is omitted the mean value of specific protein for the fasted rats drops to 0-245, the
standard deviation to 0-055 and the difference between fasted and unfasted
becomes significant (at P = 0-05), although it is now only 19 per cent. It seems
reasonable to assume that fasting produces an increase (of about 20 per cent
for this degree of fasting) in the specific protein of C.B. rats.

Fed ad lib
Fasted
P .

Table IV.— The Effect of Fasting Upon the Level of
Specific Protein in the Serum of 8 week old 3Iale C.B. Pats

Specific protein Total serum protein
(anits/mh) fg./lOO ml.)

Mean S.D. Mean S.D.

0-205±0-036 . 5-96±0-26

0-303+0-209 . 5-75±0-28

0-1 . 0-1—0-05

Bat weight
(g-)

Mean S.D.

263-8±9-3

247-7±I7-0

< 0-01

Number

rats

SERUM PROTEIK ASSOCIATED WITH TISSUE GROWTH

r)2n

Tn order to see how quickty the specific protein decreased in tlic scruin after
feeding a hatch of 27 mile C.B. rats, 9 weeks of age, were tested in (he following
Sy. At 9.30 a.m., and before they were fed, 9 of these ^

randomly for bleeding. Pood was then given to the remainder and 21 lioiiis l it
(noon) another randomly selected 9 were bled. T he remaining 9 were bled -1.,
hours after feeding. The values for the specific protein in the three groups wcic
0-983 -L 0-108 0-231 ± 0-032, and 0-240 ± 0-038 respectively. Although there
was a top in the level of about 20 per cent at 2i hours after feeding, this difTercncc
was not significant (P = 0-2). Nor were there any significant difTercnces m
total serum protein.

Effect of 2 )r&gnancy

The effect of pregnancy on the specific protein and total protein (both deter-
mined on the same sera) is shomi in Table V. Also included in the table are dat a
from rats which had just littered by a few liours (22 -f 1 days). It will be seen
that the specific protein increases steadily up to the day of delivery (22 days),
both absolutely and in relation to the total serum proteins. The total protein,
on the other hand, increases for a while and tlien undergoes a significant drop
(about 20 per cent) from the 17th to the 22nd daj' of pregnancy (P = 0-01), and
it is interesting that the specific protein does not seem to be affected Iiy this.
The higher level of specific protein attained after deliver}' (22 -j- I days) than
before may be an effect of the birth upon the tissues of the mother. The level
reached just before delivery (22 days) is about 2i times the normal female level.

Table V. — Level of Protein in the Serit7n of Pregnant C.B. Bats, Showing the
Means, Standard Deviations and Number of Sera (in Parcnthc.scs) Used

Days

pregnant

22 + 1

Specific proteins
(units/ml.)
0-449 + 0-077 (13)
0-G32 + 0- 115 (10)
0-845 + 0-251 (10)

0- 882 + 0-174 (10)

1- 053i0-291 (15)
1-301 + 0-337 (10)

Total protein
(g./lOO ml.)
f>-21i0-18 (13)
6-2G+0-30 (10)
C-42 + 0-49 (10)
G-40+0-40 (10)

5- 29+l lC (1.5)

6- 18 + 0-4! (10)

Specific protein
Total protein
7-2
10-1
13-2
13-8
19-9
21-1

100

17 dag foetal rats

Table VI summanzes the results of studies on the serum of 17 day C.B. foetuses

Th?nm?f ^ pool of either the male or female sera of one litter

The male foetus of this age has 37 per cent as much protein in its serum as its
mother and only 31 per cent as much specific protein. Both mother and foetus

2-2 day foetal rats

Table VII shows the results for 22 dav male f D i ,

The sera of individual foetuses were tested The i '"others,

what in total protein over the maternal spi-nin + ft gained some-

m bM„ a remarkable increase to tile H day Cage but tbere

i-cr degree to the mother. The foetal sLm aSll S Zot c™e:;t“t1o‘„'’of

530

D. A. DARCY

the specific protein than the maternal but this protein is now much higher reh-

tively thari m the maternal. It would be of interest to knoiv what developmenhl
event is related to this increase. ^ uieiudi

Table YI.— Level of Protein in the Serum of C.B. Pat Foetuses and Their Ihthers
on the iWi Day of Pregnancy. Means, Standard Deviations and Number of
Sera [in Parentheses) are Shown ■'

Litter
A Mother

Foetuses (male
B Mother

Foetuses (male)

C ^Mother

Foetuses (male)

„ (female) .
D Mother

Foetuses (male)

„ (female) .

AU mothers
All male foetuses

Male foetuses
Jtothers

Specific protein
(units/ml.)
0-88

0-29 (5 pooled)
0-85

0-20 (3 pooled)
0-94

0-304 (5 pooled)
0- 335 (6 pooled)
0-72

0-20 (10 pooled)
0- 17 (0 pooled)

0-848±0-093 (4)
0-203±0-045 (4)

X 100 = 31%

Total protein
(g./ml.)

Mothers ' 6-59^0-46(3)

Male foetal sera 2-43±0-20(3)
Male foetuses _

Mothers

Specific protein
Total protein

iVIothers Male foetuses

12'85 lO'S

Table VII. — Level of Protein in the Serum of C.B. Male Rat Foetuses and Their
Mothers on the 22nd Day of Pregnancy. Cleans, Standard Deviations and
Number of Sera are Shown

Litter
A Mother
Foetuses
B Mother
Foetuses
C Mother
Foetuses
D Mother
Foetus
E Mother
Foetuses

Specific protein
(units/mil.)
1-08

0- 548±0-OC9 (4)

1 - 00

0-56 ±0-132 (3)

1-00

0- 633±0-109 (4)

1- 17
0-464

1-13

0-967±0-085 (8)

Total serum protein
(g./lOO ml.)

Mothers 5-67±0-6f) (3)
Foetuses 2-52±0- 18 (18)

Foetuses

Mothers

X 100 = 44-3%

Specific protein jqq
T otal protein
Mothers Foetuses
19 39

All mothers
All foetuses

Foetuses

Mothers

1-076±0-076 (5)
0-730±0-219 (20)

100 = 08%

It will be noted that only 3 mothers w^ere used for the total protein calculation.
This w^as because the 18 foetal sera w^ere entirely dratvn from the litters of these
three (C, D, E). It is of some interest that there appeared to be a rough pro-
portionality between the number of foetuses in the 22 day pregnant mothers and
the level of specific protein in their blood. This was not investigated further.

New born rats

Table VIII shorvs a comparison between the blood proteins of C.B. mothers
and their offspring a few hours after birth (0-day old). The individual sena ol
the young W'cre examined. There has been remarkably little change as a resu
of birth. The offspring have a lower concentration of the specific protein ttian

531

SEBUM PBOTEIK ASSOCIATED WITH TISSUE UBOWTH

Number of Sera Employed- {in Parenlheses) Are Gmtji

Totnl spnitn i)rol<'in
(g./lOO nil.)

Mothers fi- 18i0'41 (10)

Mnle offspring 2-f)ti0'20 {—>)

" ■ 2-r)3-i-0-18 (3r>)

Litter
Jlother
Male offspring
Female offspring
Jlother
jMale offspring
Mother
Male offspring
Mother
Male offspring

All mothers
All male offspring

Specific protein
(units/ml.)
1-30

0-803i0-072 (0)

0- 82G±0-0S8 (5)

1- 05

0- 04 ±0-117 (5)

1- 46

0-835±0-071 (6)
M6

0-G42±0-063 (5)

l-243±0-n7 (4)
0-80G±0-129 (22)

Female offsiiriiig

Male offspring
.Mothers

X inn i3<;

Specific protein
Total protein

X 100

Mothers

20-1

Male offsiiring
30- fi

Male offspring ^ jqq ^ ggo/^
Mothers

Colostrum and milk -n-ere obtained from motliers by the technique of Lucltey,
Mende and Pleasants (1954), in order to see if the specific protein tvns present.
Sufficient amounts of colostrum for quantitative tests tvere obtained from two
rats. In one the specific protein, though present, tvas well under 0-2 nnits/inl.,
while in the other it was found to contain 0-15 units/ral. (± 7 per cent). A sample
of milk obtained 10 days after deliverj’-, from another rat, contained about 0-()!l
units/ml. It should be mentioned that about fi'’® of tlie 0-day old rats Iiad
colostrum in their stomachs at the time of weighing and bleeding.

Certain other points in the table are of interest. The difference in specific
protein between the male and female offspring in litter A is not significant (P =
0-3), but the 0-day males in general had a higher total serum protein content than
the females (P = 0-05). The 0-day males were also heavier (P = 0-05) than the
0-day females (6-90 i 0-54 g. compared with 6-65 ± 0-G4 g., there being 40
animals in each group).

DISGUSSIOB

This quantitative study of the rat serum a-globulin, previously found to be
associated with tissue grovdh, has confirmed the earlier semiquantitative results
The protein has been shmvn to reach a high level in foetal and young rats and in
pregnant females, compared with the normal adult level.

The normal adult level is about 0-24 units per ml. of serum for male C.B. rats •
there is a steady deeline to this level from a maximum value of about 0-87 at 1 u-eck
0 age. The growth rate of the rats can be shoum to decline in a similar way the

mt, arc usually coSetd to be "S” LTff^r “'’f f •“*

tbe definition of this stage '“"""Ction with

Other rat strains tested gave ualucs for S rveeh old males closely appro.vi-

532

B. A. DABCY

mating tiiat for C.B. males. Since groAvth rate of the C.B. rats ivas considerablv
higher than for these other strains this raises a difficulty for the hypothesis tint
the specific protein is directly concerned with growdh. It may be, however that
the level of the protein in the serum need not be exactly proportional to the tr^owth
rate especially betiveen different rat strains. The results indicated, moreover
that the G.B. rats had a slightly higher level (about 10 per cent) than the slow
growing August rats, a difference wliich might prove significant if large numbers
were tested.

A surprising finding was that the 8 week old female C.B. rats showed a level
of the protein which was about SO per cent higher than the males. This is an
important clue as to the role of the protein. On the growth hypothesis it could
be argued that tliis reflected the higher growth activity in the female reproductive
system. ^ A study of the female during the oestrus cycle ivould test this idea.

During pregnancy the level of the specific protein in the mothers’ blood
increased steadily vdth the growth of the foetuses and at birth reached a level
three times as high as in the normal female. It is too early to speculate about the
reason for the increase, beymnd noting its association ivith foetal grmvth. It is
of considerable interest that the 20 per cent drop in total serum protein of the
mother which occurs betu’een the 17th and 22nd day of pregnancy did not affect
the level of specific protein in her blood. This drop, moreover, was not accom-
panied by a corresponding increase in the total serum protein of the foetus.

Some interesting data were collected on the serum proteins of the foetal rat.
The specific protein -was low at 17 days, but by 22 days it had increased tluree-fold,
M'ith very little increase in the total serum protein. It ^YOuld he of great interest
to know what developmental event is associated with this increase. It might
represent a sudden increase of selective permeability of the placental membranes
to the specific protein so that it can enter more freely from the mother’s blood,
or it might represent the starting up of the foetus’ own synthetic machinery for
this protein. Birtli made remarkably little difference to the foetal serum proteins
and it is a matter of speculation whether the specific protein found in the colo-
strum and millv was absorbed undenatured across the gut of the young rat. The
low level of the specific protein in the serum of 1 7 day foetal rats is another fact
which appears to disagree with the hypothesis of a direct association between the
protein and growth, for the 17 day foetus must be growing very rapidly indeed.
But this need not be a disagreement, for the level in the blood represents an
equilibrium between inflow and outflow, and either might be abnormal in the 17
day foetus.

It was stated in the introduction that the specific protein most closely resem-
bles, of aU the serum proteins, the group of globulins called “ fetuin ” which are
believed to be characteristic of foetal protein. The evidence on which this is
based Avill be presented in a later paper. But it is supported by tiie present
finding that both are high in the foetus and young animal. HoAvever, fetuiu
reaches its peak in the foetus (or the calf) ivhile the present protein appear to
reach its peak at about a rveek after birth. Furthermore, the present specino
protein, unlike fetuin, reaches a high level in pregnant females. These differences,
however, may be due to the different species tested. ^ _ fron-

Fasting plar^s a significant role in the level of the specific protein. ‘

to Avhat might be expected it causes an increase in the levml, this varying i
about 20 to 35 per cent for a 20-22 hour fast depending on the rat strain emp o.A

SERUM PROTEIN ASSOCIATED WITH TISSUE GROWTH

This fact is another interesting lead as to the role of tlie protein. It is diilicult (o
reconcUe it ^rith the hjTothesis of a direct association between I™
ceU division, for fasting is known to cause a decrease in mitotic act ivit> at son oral
sites in the body (Leblond and Walker, 1950). It siiggc.sts a relationsliip between
the protein and stress, an hj’^iotliesis which could be easily tested.

SUMMARY

1. A study is presented of a rat serum a-glolnilin wliicli Iiad previously been
found to increase considerably during tissue growth, whether normal or ncoiilastic.

2. The level of the protein in the serum was measured by means of a simple
immunological method developed for the ]mr])ose.

3. The level of the protein Nvas folloNved in the male C.B. rat. It was com-
paratively loNV in the serum of the 17 day foetus, but increased rapidly thereaft er
to reach a peak about a week after birth. From there it declined sharply to a
low level at about S NN’eeks of age.

4. The adult female C.B. rat had a level of this protein nearly twice as high as
the male. During pregnane}’’ it increased steadily, reaching nearly 21 times
the normal level just before birth. The protein was also ]>rcsent, though in low
concentration, in the colostrum and milk. The relation betwen the foetal ami
maternal serum protein Nvas studied.

5. The 8 Nveek old males of three inbred lines of rats (Wistar, August. Marshall)
had a level of the protein in their serum Nvhich differed only slightly from that of
the C.B. stock.

6. Fasting caused a significant increase in the protein.

7. The possible role of the protein is discussed.

I Nvoidd like to express my thanks to Dr. D. J. R. Laurence for valuable
assistance in obtaining a purified preparation of the jirotein.

This investigation has been supported by grants to the Chester Bcattv Re-
search Institute (Institute of Cancer Research ; Royal Cancer Hosiiital) froiii the
Medical Research Council, the British Empire Cancer Campaif^n, the Jane Coflin
Childs Memorial Fund for Medical Research, the Anna Fidlcr Fund and the Xal-
lonal Cancer Institute of the Rational Institutes of Health, U.S. Ihdilic Health
Service. ’

REFERERCES

CAJrPBELL, P. R., KekxOT, B. a. AXD RoitT, I. M — (1959) Biochem T 71 In-

USAF. Report Ro. 32. ’ ' J/w/icmc.

Laubexce, D. J. R. — (1956) Biochem. J., 62 36 P

*.■„ 36. 255.

Mivers. ff . jf. Deukot H r^^-w f 5^. 3«-

OOCHTBEEOKY. 0. (1948) Ark.' lit el 7o "■

534

A QUANTITATIVE STUDY OF A SBBUai PEOTEIU ASSOCTATli'n

SfJ™ m m Sm-

D. A. DAECr

From the Chester Beatty Research Institute, Institute of Cancer Research,

Royal Cancer Hospital, Fulham Road, London, S.W.3

Received for publication July 19, I960

In the preceding paper (Darcy, 1960) a study was presented of a rat serum
a-globulin, giving its level in the normal animal at various ages, and the increase
in this level which occurs during pregnancy and fasting. The main purpose of the
present paper is to compare these results with the levels reached during tumour
groudih. It was hoped to obtain, at the same time, some hglit on the biological
role of this protein which appears so closely related to tissue grovdih and regenera-
tion (Darcy, 1957).

BIATERIALS AND METHODS

Animals. — Male rats were used tlmoughout and, except where otlienrise
stated, were of the C.B. stock preUously described (Darcy, 1960). The inbred
August strain rats irere used for transplantation of two tumours which arose in
that strain. Transplantation was performed bj^ means of a trochar tlirough a
1 cm. incision in tlie flank wliich was closed with a single Jlichel’s clip. The C.B.
rats used for transplantation were between 6 and 8 weeks of age. BenzpjTene
sarcomas rvere induced by similarly implanting a small pellet of the carcinogen
and waiting 4 to 6 months. Butter yellow hepatomas were induced hj feeding this
substance (4-dimethjdaminoazobenzene) as 0-06 per cent of the diet (which
contained 20 per cent protein) ; the tumours appeared in about 8 months. Bleed-
ing was performed under the conditions described in the preceding paper. All
operations were carried out using ether anaesthesia.

Measurement of serum protein . — Both the specific a-globulin and the total
serum proteins were measured by the methods described in the preceding paper.

BESUI-TS

Walker tumour

The growi;!! of this tumour in the C.B. rats was extremely rapid and was
usually lethal in 3 rveeks. Tv'o experiments with tliis tumour are shown in Fig. 1-
In each experiment a batch of 12 C.B. rats of the same age was implanted at one
session, and at the indicated intervals thereafter two rats from the batcii were
bled and their tumours removed and rveighed. T\vo of the 12 rats were kept as
controls : in the first experiment they rvere untreated and bled at 1 and 4 days ,
in the second experiment they were sham-operated, an empty trochar being
inserted, and both u^ere bled at 1 day after the operation.

A SERUM PROTEIX IN TUMOUR-REARING RATS

iiijr)

The first point of interest in the graphs is that the level of the specific protein in
the serum of the tumour-bearing rats was already about three and a half times t he
level of the controls at 24 hours after the operation. Sham-operation of the
controls did not appear to have anj' effect (the normal level for these rats is aliout.
0-24 units/ml.). Trom 24 hours onwards the specific ])rotcin increased in the
tumour-bearing rats, although at a reduced rate. The rate increased again st arting
at about 8 daj^s after transplantation, by which time the tumours weighed bet ween
15 and 36 g. The level of specific protein reached at 12 days after trans])lantal ion
was 21 times that of the controls in Experiment 1 and 14 times that of the cont rols
in Experiment 2.

While there is a general correspondence between the size of the tumour aiul
the level of the specific protein in the blood, it is not a close one. This can be
seen from the shape of the two cuiwes in Exjieriment 1, and from a coin])arison

taie thi '’TT of the

the animal: a more likely candidate ^is the the tumour in

more probably, of actively dividing cells for the^ ^ u^ tumour cells or,

massive necrosis of their centres and only a cortex l>ad a

It IS instructive to comnare th^e.^^ ^ of viable cells.

the tumours grew somewhat faster up to Thus, while

^le specific protein remained consiVfprfm i ^ Expenment I, the level of

From 8 to 12 days there - E.xperiml f

. ccompa„,edbyat„„„„rg™,^,htJ'^^JJ®So in Experiment 1

of ■ rats witli large WalkS t^mT 'loterminations were made

^or the specific prot~S“ prjI^T^f ^

' ’ agrees well with the

536

D. A. DAECY

experiments The mean value for total serum protein wa.
4-64 g./lOO ml. (as compared with a value of about 5-8 for healthy Limals of
prece^ng paper). But the most strildng value was the ratio of speoific/toHI
protein (xlOO). This was 69-9, compared vdth the normal value of about f'o
Ihe Jnghest value encountered in any normal animal was 30-5 in the new-boni

TQ,tf0

Ascites Walker tumour

The Walker tumour can be made to grow successfully in an ascites form. It
adheres mainljr to the omentum and mesenteries of the peritoneal cavity where it
forms clusters of tumour nodules. But tumour cells also float freely in the ascitic
fluid. Table I shows the results of testing the serum and the supernatant ascitic

Table l.~Levels of (he Specific Protein and Total Protein in the Ascitic Fluid and
Serum of Male C.B. Rats Bearing the Walker Ascites Tumour for 7 Dags

Specific protein Total protein

(units/ml.) (g./lOO ml.)

Rnt

Serum

Ascites

Senun ^ ^'>0

Serum

Ascites

Serum

O-QO

0-57

3-65

2-95

O-So

0-23

2-95

2-60

0-34

0-22

2-95

2-60

0*55

0-32

3-65

2-95

0-09

0-41

3-65

2-95

0-42

0-2G

4-16

2-95

1-08

0-70

3-65

3-30

0-77

0-20

6-07

5-72

0-48

4-33

2-95

1-01

1-05

4-16

3-65

1-33

0-67

4-33

3-12

Average

0-835

0-465

56%

3-90

3-25

SI?t

fluid for the specific protein. The rats (11411011 were S weeks old) had been inoculated
intraperitoneally with the tumour cells 7 day^s before. It should be noted that
the average value of the specific protein in the serum of normal S week old male
C.B. rats is about 0-24 (cf. preceding paper). It will be seen that this value has
been more than trebled, on the average, in the serum of rats bearing ascites
tumours. But in the ascitic fluid itself it has only been about doubled. The level
in the fluid varies from 26 to 66 per cent of that in the serum of the same rat.

This observation has an important bearing on the site of origin of the specific
protein. It strongly suggests that the protein is not produced by the tumour
(udiich might be expected to secrete it into the ascites) but at some distant she
in the body whence it is carried by the blood. This is supported by’^ the fact tha
the ascites was extremely bloody (it was indistinguishable in colour from who e
blood) and that the ascitic supernatant contained on the average 81 per cent as
much protein as the serum. But since the specific protein reached a level m ic
ascitic fluid which was only’" 56 per cent that of the serum, this suggests the possi
bility that the specific protein is being selectively withdrawn from the fluid y
-tumour cells. When the ascitic supernatant was tested on the Oucliter on}
diffusion plate it appeared to contain the same complement of proteins as i
serum of the same animal.

A SERUM PROTEIN IN TUMOUR-REARING RATS <

The low total protein content of the serum of these rats is notcwortliy, lieiiifi;
only 3-96 e./lOO ml. compared with 4-64 for the 12-14 day old solid Walker tumours.
The ratio of specific protein to total protein { X 100) is also low compared with tha(.
for the sohd tumours, being an average of 21-0 for tlie scrum and 14-3 for the ascil ic
fluid Nevertheless it is remarkable that at a time when t!ie total serum ])rotein is
so seriously depressed the specifle protein should have increased so iiuich above
the normal level. The actual weight of tumour in these ascites-carrying rats was
small, not more than 10 g. at the most ; but there was also little or no necrosis.

Analysis of the jelly surrounding the Walker tumour

It has been suggested that an increase in the serum glycoproteins may result,
from a depoljTnerization of the ground substance of the connective tissue, giving
rise to smaller soluble proteins which leak out into the blood (Catchpole, 19.")0).
This hj'pothesis might reveal the site of origin of the jwesent iirotcin, so it. was
tested in the foUo^ving way.

The Walker tumour groving subcutaneously in the C.B. rats ]iroduces a
considerable quantity of a clear water}' jelly in the connective tissue around
itself. When this material is excised and centrifuged it yields a white sediment
(connective tissue) and a clear supernatant. The supernatant was analyzed for
specific protein, total protein, and also for the number of individual senim jirotcins
it contained by means of the Ouchterlony gel diffusion test. This last tc.st., employ-
ing rabbit antiserum against serum of Walker tumour-bearing rats, showed thiit.
the supernatant contained the proteins of serum, and in approximately tlie same
proportion to one another, vith the exception of certain higher molecular weight
proteins which appeared to be in relatively lower concentration than in the seriun.
These larger proteins could be detected by the convex curvature of their lines
(Nomgold and Van liceuwen, 195/). Such an effect might be predicted .since the
proteins presumably get into the jelly by diffusion and there may even be some
filtration. Apart from this difference the jelly supernatant appeared to be a dilute
form of serum.

Table II.-

~The Protein Content of the Liquid Phase of the Jelly Surrounding the
fl allxr Tumour Compared with that of Serum

specific protein
(units/ml.)

Rat Jelly Serum

2-70

2-64

0-19

0-29

0- 03

1- G7
0-60

4-40

4-lG

0- 30
0 -

1- 42
3 -.59
1-84

■8

1-95 4-02

AvernRe .

2-4.7

1-4G

4-14

2-74

Total protein
(g./lOO ml.)

Tumour

X 100

Jelly

.Serum

.Telly
Serum ^

2-60

5-60

2-95

4-33

2-60

4-16

2-60

2-77

4- 33

5- 21

3-65

5-90

2-77

2-60

106

2-43

4-51

2-95

5-03

53% .

2-81

4-63

61%

Size Conclitic

.Small

,, Goml.

Necrolie.

Large Mninly

„ good.

Huge Part good,

part ne.
erotic.

*» Necrotic.

638

D. A. DAECY

Table II shows tliat the jelly contained on the average only 53 per cent as
much of the specific protein as the serum, and Cl per cent as much total protein
It is unlikely therefore that the jelly is the site of origin of the increased specific
protein in the serum. There is again a case for the argument that specific protein
is being selectively taken up from the jelly by the tumour. Certainly the specific
protein was lowest relative to the other proteins in the jelly of the two tumours
(rats 4 and 7) which showed least necrosis and v'ere in best condition. No e.’cplana-
tion can be offered for the case in which the specific protein was in higher relative
concentration in the jelly than in the serum (rat 1). In the case where the jellv
fluid appears to have a higher protein content than the serum (rat 7), the difference
is probablj^ not significant and the extraordinarily low protein content of the serum
(2.60 g./lOO ml.) may reflect the advanced state of the tumour growth in this
animal.

The Auguat tumour PWA.2

This is a transplanted tumour which originated as a mammary' carcinoma
in the inbred August rats in wliich it gives 100 per cent “ takes ”, Its interest
for the present work is tliat, in contrast to the Walker tumour, it is vei^^ slow-
growing and shows little or no necrosis. Ten August males were implanted with

Fig.

-Increase of the specific protein in the serum of August rats at 16 daj s (no tumour)
and 73 days after impfantation of the PWA. 2 caremoma.

the tumour and bled as follows : two at 16 days after pfting two at 37 days,
and the remaining six at 73 days after grafting. The results are

At 16 days after grafting there was no measurable tumour and , .

level of the specific protein above the normal for these rats (about 0-^ u
Darcy, i960). At 37 days after grafting the two ammals bled gave ^

and 0-29 for the protein. Unfortunately Thev ave not

measured (the length and breadth measured through the skin). T1 j

A SERUM PROTEIN IN TUMOUR-REARING RATS

h'M)

therefore sho^ni on the graph. But ^vhcn all the tumour si/.c.s vyorc expre.ysefl as
the product of length X breadth, the two 37 day points straddled the line of
regression just below the smallest of the 73 day tumours and did not disturb i(,s

The results show that there is a close relationship between tiimonr si/.e and
level of specific protein, much closer than was shown by the A\'alkcr timionr.
These August tumours had only a small amount of necrotic-Ioolcmg ti.ssue m tlieir
centres while the Walker tumours were widely necrotic except for a cortex-. I his
suggests that the level of the specific protein is proportional to (be mass ol living
tumour tissue and supports the concept that the basic relationship is bet ween t he
protein and the mass of actively dividing cells.

It could be objected against tins interpretation of the results that the increase
of the specific protein in the serum is simply a function of time after iiiocnlation
and may not be a function of the actual size of tlie tumour. To test, this jiniiit.
the coefficient of correlation was determined for the 0 sera and tiimoiirs which
were taken on the 73rd day after transplantation. Tliere was found to be a
significantly positive correlation between the level of the specific jirotcin and the
tumour weight (r = 0-9073, P = 0-02 — 0-01), showing that the relationshi]i
does not depend on the time of residence of the tumour.

The total serum proteins for the rats bled at 16, 37 and 73 days after tumour
inoculation averaged 5-72, 5-72 and 5-17 g./lOO ml. respectively and the ratios
of specific to total protein (xlOO) were 3-15, 7-43 and 21-4.

The August osteosarcoma D.lll

This is another tumour which arose in and is transplanted in the August, rats.
It was studied partly in order to have a sarcoma to compare with the PWA.2,
and partly because it is exceedingly fast-gror\-ing compared with tlie P\\'A.2.
Eleven August rats 7 weeks of age were implanted nith the tumour and its growth
was followed by measuring the lengtli and breadth with calipers througli the skin.
By ten days after transplantation the mean size of the tumours was 0-93 cm.=
(product of the two measurements). On the 1.5th, IGtli and 17th days after trans-
plantation the mean sizes were 5-95, 7-0 and 8-8 cm.2 respectively."^ The animals
were bled and the tumours weighed on the 17th daJ^

The results are shornr in Fig. 3. They are completely contrarj^ to what was
expected on the basis of previous experience, and appear at first si^ht to refute
the hypothesis that the specific protein is related to tissue growth Since the
osteosa^rcoma is so much faster-gron-ing than the mammary^ carcinoma P\VA.-’
it might be expected to cause a much liigher level of the snecific -nmipin in fi, '

r»- » 0-365 unit/mVrS muf LCT,; H,a

for the PB A.2 tumours at 73 days (where the average was Ml unite; /ml i , i '•

betSS\rreo^

there is a slight tendency in the onnosite rlironf • ® P™tein. Indeed

the lower theVel of spedfie proteSee^^^ humour

not significant on the present^ample, however (r = ^0 If P '"‘'^fionship was

It was slightly improved when the estimated necrotic fJ?’ ^ ~

first subtracted from its weight (r = —0-288 P == q 3)°^^^^ * tmnour was

540

D, A. DABCy

These results cast doubt on the hypothesis that the level of specific protein in
the serum IS always positively related to the total amount of tissue growth I'n"
on m the body at a particular time. They do not, however, refute the InmoLis
that the specific protein is itself concerned in tissue growth, for it is possible that
the rate of growth of the osteosarcoma is such that the specific protein in the serum
IS removed as fast as it enters, so that only a relatively low level can be maintained,

l-Oi

0 - 8 |
c

'~S

io-6

20-4i

io-2

S, !

OT I

10 15 20 25 30

Weight of tumour (g.)

Fig. 3.— Specific protein in the serum of August rats 17 days after
implantation of the r).177 osteosarcoma.

Furthermore it is known that August rats have a low capacity for protein s3mthesis
and growth compared vdth C.B. rats (Elson, 1958).

Table III. — The Level of Protein in the Serum of August Eats Bearing the D.lTi
Osteosarcoma for 12 and 15 Days. Means and Standard Deviations Shown

Days after
trans-
plantation
12
15
P

Kats

Tumour weight
(g-)

7-98±3-77

22-(>0±4-55

<0-001

Total serum protein
(g./lOO ml.)
G-00±0-36
4-40rb0-16
<0-001

Specific protein
(units /lOO ml.)
0-40I±0-II6
0-G53±0-057
O-Ol-O-OOI

To examine this question further, a batch of ten rats with osteosarconitis
were examined, five being selected randomly and bled at 12 daj's after
plantation, the remaining five being bled at 15 days after transplantation. >
results are showm in Table III. The tumour grew faster than in the previous expen-
ment ; it had approximate^ trebled its weight during the 3 day interva .
levels of specific protein in the serum were higher than in the ’

but were still lower than might be expected from the size and grmvth rate oi r
tumour. An important observation is that the level was higher at the later • o

A SEBUM PROTEIN IN TUMOUR-BEARING RAM’S

n 1 1

of tumour grouth, suggesting that there tvas, in this experiment, a ,H»itivc rein-
tionship between tumour size and level of speeitic pi otein.

The^effect of necrosis could be examined in tins tumour, where its txtenl uas
estimated and foimd to vaiy from 0 to about f of the tumour voluiiic No re la lon-
ship was found between the estimated weight of necrotic inatenal and tiu I{\cl
of the specific protein in the serum. No relation.ship was found between tninoui
size and the extent of necrosis.

Induced hepalomas

These tumours were induced in the C.B. rats by feeding them the azo «he,
butter yellow, in a 20 per cent protein diet. Tinnours ap])earcd after alioiit S
months and, where necessary, were confirmed histologically as being hepatomas.
The results of testing the sera are shown in Table where they arc grouped
according to the approximate size of the tumours.

Table IV. — The Level of the Specific Protein in the Serum of Pats Bear! nr/ Ilcpatomas
Induced by Means of Butter Yellow Feeding

Tumoiu-

Specilic,

jirotein

Rat

Size

Condition

Metn.stn“e.s

(g./I(*0 ml.)

. No tumour

—

None

0 - 2 :,

. Small

Good ; liver normal

»• •

0-41

Good

• » •

0-.M6

. Moderate

1-2

. Large

Partly necrotic

—

2-4

• if

Good. Liver abnormal

I-O

. Verj- large

CX'stic and a little necrotic. Much

1-.'.

ascites

• if ff

Half necrotic. Ascites

2- 6

‘ if if

Mainly good

2-6

• ft if

Good

, - -

.M- 1

• iff if

Good. 40 g.

None

2-. 7

• ff if

Partly cystic and haemorrhagic,

Some

1-.-.

• f if

15 g., good

Necrotic. Good metastases

Much

2-8

■ ff if

Partly cystic

ff ff

Cystic and necrotic, but metas-

Verv much .

7- 6

■

tases good

Good

, (40 g.)

Verj- much .

3-8

(30 g.)

The first rat in the table had noturaourafter lOj monthsof butteryellowfeediiin
Its serum level of specific protein was normal. There is a general correspondence
between the size of the tumours and the level of specific protein The hif'Iicst
titres were found in rats with metastases. VTiere there is no entnr ° f ^

Stases in the table they were either absent or very slight Rat 1 5^avp flip I

title ever recorded in this laboratorv • its rvr;m!n-J+ ^ highest

but the metastases were vast ; it had beeiTferthe diefor' necrotic

animal ivhich was excludpfl frnm fLo f li i the (i3e for only / months. One

but a fibrotic tumour appa^Sl devl® “ Jiepatoma

titre of 4-2. ’ developed from a cholangioma, gave the higli

.cm th.f they „.y he eompHcated by liver d.mage.Vthem is"

542

D. A. DARCY

(as results to be presented in a later paper rviU sliow) that the specific protein k
the liver. This might explain, for example, why the specific protein
failed to rise with small tumours (rats 2 and 3) to the extent it did with smaU

allrer tumours, but difference in growth rate of the tumours could also account
for this.

The sjjecific frotein during carcinogenesis

The foUoiving experiment shows the effect of another type of induced tiinionr,
this time a sarcoma, upon the level of the specific protein in the seriini, and also
the effect during the genesis of these tumours. C.B. male rats were implanted
subcutaneously irith small pellets of 3 ; 4-benzp3Tene. Sarcomas usually arise in
about 75 per cent of these animals at the site of implantation (and usuall3’^surroiintl-
ing the pellet, which is not absorbed). Seven such rats were bled from the heart
at monthty intervals, and the serum level of the specific protein measured. The
results are shown in Table V.

Table V. — The Effect of Carcinogenesis on the Specific Protein. Serufn Titres of
the Protein in 7 Rats at Monthly Intervals Starting Three Months After Thetj
Had Been Implanted Subcutaneously loith a 3 : i-Benzpyrene Pellet. The
First Appearance of the Tumour is Indicated by the Titre in Bold Type and the
Apiproximate Tumour size (in cm.^) in parentheses

Specific protein (units/ml.) in serum

C months 7 months
0-22 3-35 (2o-S)

Bnt 3 inontlis

1 . 0-18

2 . 0-21

3 . 0-29

4 . 0-44

5 . 0-78

G . 0-47

7 . 0-44

4 months

5 months

0-21

0-43

0-42 {3-2)

0-40

0-35

0-30

0-27

0-30

0-47

0-40

3-60 (25)

3-84 (20-4)

0-31 2-84 (56-0)

0-23 0-32

0-43 O-M(i-O)

0-25 —

Two of the rats (4 and 6) did not develop tumours during the period under
observation. Their serum level of the specific protein remained well within tlie
normal range (0‘245, S.D. i 0-069 ; cf. Darcy, 1960). Of the remaining five rats,
two (2 and 7) had tumours when examined 5 months after implantation of the
carcinogen. For rat 2 the tumour was still rather small (3-2 cm.^) and the specific
protein low (0-42), while in rat 7 the tumour was alread3j large (26 cm.^) and the
specific protein high (3-60). At six months the tumour in rat 2 had grovn con
siderably (20-4 cm.^) and the specific protein had increased correspondingly
(to 3-84). By 7 months the three remaining rats had tumours ; in rat 5 the
was small (1 cm.®) and its specific protein only 0-44, whereas in rats 1 and 3 the
tumours were 25-8 and 56-5 cm.® respective^ and the specific protein gw

2-84 respectively. . , , i t „„rc

Two main conclusions can be drawn from these results. The first is tliat tumour
of smaU but quite important size, in relation to the host’s size, can exis in lo
body vdthout causing the level of specific protein in the serum to rise appreciao 3.
In short, for this particular host-tumour situation the level of specific pro e
the serum would be useless as a diagnostic tool. It is interesting to ^ » j

wdth the Walker tumour in the same animals, where the titre increases 3 0

A SERUjr PROTEIN IN TUMOUR-HEARING RATS

Mithin a day of transplantation. The difference may lie in the (linei ont sliced f
groArth of the two kinds of tumour, although tins may not lie ( he only cau.se.

^ In the period before tumours appeared the level of the "

the serum Avas higher than in normal rats. For example, at .1 mont hs after imph i -
tation the average Avas 0-40 units/ml. compared Avith 0-245 in the normal rat..
It Avould be rash, hoAvever, to ascribe this to the carcinogenic proces.s, for such an
increase might he produced by many stresses, c.g. slight overcrowding m the cages,
mild infections, the heart puncture, etc. Furthermore, rat 1, seems to have
developed its tumour Ai-ithout any sucli preliminary increase.

The second conclusion is that the specific protein in the seriim inci eases
greatty A\dth a second tjqie of induced tumour, this time a sarcoma. 1* nrthermoro,
the increase is again roughty in jiroportion to the size of the tnmonr, alt hough
the fit is by no means exact. An exact fit Avould not he exiiectcd because avc arc here
dealing, not AA-ith a single homogeneous tumour like the August PWA.2, but Avit li a
group of independent sarcomas each Avith its OAvn gi-OAvth rate and other character-
istics. The mean size of the 4 large tumours in the table Avas 32-2 cm.- lloiibling
this quantity ghms the approximate AA'eight in grams. The mean specific protein
IcAml of these four tumours Avas 3-41. This result is comparable Avith that for the
Walker tumour.

The total serum proteins Avere determined for the blood of other rats bearing
large henzpjTene sarcomas, aA^eraging about 40 cm.- The average value was 5-liI
g./lOO ml. Avhich is relatively high compared Aiith that for adA’anced Moniker
tumours (4-64). The A’alue in normal rats is about o-S. The ratio of specific
protein to total protein AA-as A’-ariable, for e.xaraple in three rats AA-hose tumours
measured 15-4, 19-5 and 20-7 cm.^, the specific protein AA'as 0-90, 1-01 and 4-20
respectivelj’^ and the ratios of specific to total protein (xlOO) AA-ere 17-3, 19-4 and
66-4 respectively.

The specific protein and the “ K ” lines

In a comparison of cancer serum AA-ith normal serum in the rat (Darcy, 1955)
using the Ouchterlony gel diffusion teehnique, it Avas reported that the ’cancer
sera could be distinguished by the presence of precipitate hands Avhich migrated
ahead of the albumin band. These bands Avere called “ K ” lines. They Ai’ere best
seen Avhen the antiserum was against normal rat serum. In the present study it
was found that one of these bands Avas eaused by the specific protein ; furthermore
It Avas the most prominent one and the one given by most antisera. The K line

w interpret : the higher concentration of the

specific protein m the cancer serum causes its band to move from its normal

?s Sualjy"thTleaX7or for ntmXe^^^^^

S tSSod

JJiscussiojr

of semi-,oant,t,«ve finding

onJer study. The nonuTl aS lev^SsS
0.. fu, O.B. „a,e nets ,ag

544

X>. A. DARCY

jughest JoA^el encountered for normal males was 0-87 at 1 week of a^e • the hialiP^t
leAml m females AA'as about 1-2 in the last stages of pregnancy, ^^^ith the growth of
a tumour levels of beWeen 3-0 and 4-0 AA’ere common, not only for transplanted
tumours but for induced ones. Wliatever its role, this protein certainly assumes
an important position among tlie serum proteins during tissue grmvth. It was
demonstrated, in the favourable case of the PWA.2 tumour, that there was'a
significant positive correlation betn-een the size of the tumour and the level of
this protein. Tliis relationship was somevdiat masked in the case of the necrotic
Walker tumour, strongly suggesting that it is the amount of healthy tumour
tissue Avith Avhich the protein is correlated. But the basic relationsliip may be
betAA’een the protein and the total amount of groudh in the tumour and in the boclv
(i.e. the mass of groAA’ing tissue times its rate of groAAdh). For tlie only factor
common to the various situations in which an increase in the serum level of the
protein has so far been obserAmd appears to be groAidh. There are, hou'ever, one
or tAA'o situations Aidiich cannot easily he fitted into this pattern, especially the
small but significant increase in the serum IcA’^el of the protein aaIucIi occurs during
fasting. Fasting is knoAim to inhibit mitosis in seA^eral sites of the bodJ^ It may
be, lioAA'eA'er, that it increases mitosis in another part of the body to the extent
AAdiich giA'es a small nee increase OA'cr the normal total level. In an 3 ' case a correla-
tion betAA’een the serum leA’el of the protein and groAA’th could not ahA’ay’s be hoped
for since many other factors may influence the level. The case of the fast-growing
osteosarcoma D.177 in the August strain rats maj’ be an example. This tumour

caused a relatively small increase of the specific protein in the serum (contrap'
to Avhat the groAA'tli lij^potliesis would predict) and there Avas no positive correla-
tion betAveen tumour size and the protein titre, at any rate in the first experiment.
This suggested the supplementary hj'pothesis that rate of AA’ithdrawal of the
protein from the blood AA’as so great, under the influence of this tumour, that the
infloAA’ could only maintain a rather Ioav blood leAml ; in support of this is the fact
that August rats are knoAra to be relatiA'ety Aveak protein symthesizers. An
alternatiA’e explanation is that the D.177 tumour has some metabolic peculiarity.
It Avould appear, on the AA’hole, that the hj’pothesis that this protein is directly
concerned AA’itli tissue groAA’th, remains tenable eA’en though the serum titre of the
protein may not ahvays be proportional to groAA’th. It may be, hoAA'eA’cr, that
certain conditions aa’iII be found (e.g. infections and certain stresses) aa’Iiosc effect
on the protein will render the groAvth hj’pothesis untenable.

Another hypothesis, namelj’, that the specific protein originates bj’ depot} -
merization of the ground substance of the connecth’e tissue at the site of tissue
groAA’th, AA’as put to the test bj’ analysing the AA’aterj’ jelly in the connectwe tissue
surrounding the Wallrer tumour. On the lij'pothesis, a higher concentration o le
protein AA’ould be expected in the jelly tlian in the serum. Instead it Avas t la
the aqueous phase of this jellj’ contained onlj’^ 53 per cent as much specific pro cm
as the serum on the aA’erage. Another bloAA’ for this hjqiothesis is the fin mg la
Avhen the Wallrer tumour is groAvn in the peritoneal cavitj’, the ascitic fluid con ai
only 56 per cent as much specific protein as the serum on the average.

In both the above experimental situations, the jelty fluid and ascitic flm a\
found to contain all or nearly all the proteins of serum, thovigh in lower . '

tion. The ascitic fluid AA’as actually bloody, though it did not clot. The in er c
fact appeared that the specific protein AA’as present in loAver concentratio
to the total protein, in the tAVO fluids than in the serum. This strongly

1 , relative
suggested

A SERUM PROTEIN IN TUMOUR-BEARING RATS

r..jr,

tlie possibiHty tliat the specific protein was lieing talccn up liy the ^ •

selectively either from these fluids or from the serum wlucli went to form f.licm
It is unity that diffusion could explain the relatively low level of Ihe siicci ic
protein in these fluids, since it diffuses as rapidly as scrtim alhuinm in agar gels.
It may he, however, that these fluids contained a large ainoimt of some iirofeiii
which is either absent from the serum or was undetected by the Ouclitcrloiiy
method, and this would account for the difference.

Necrosis might be thought to he a factor influencing the increase of f lic protein,
especially as necrotic tumour tissue is known to cause an increase in serum
al-globulin and a decrease in serum albumin and y-glohulin (Dontcnwill, Him-/,
and Molw, 1959). It is clear, however, that it is not a necessary factor, for high
levels of the specific protein occur where there is no necrosis, c.g. in young rals.jn
pregnant females and in animals undergoing regeneration (Darcy, 19C0, 1957).
Increases also occurred unth tumours which were not necrotic, c.g. some of tlie
August PWA.2 tumours. There simpty remains the question of whether necrosis is
a contributor!’ factor to the increase in specific protein. Against this is the fact,
that homografts of normal tissue, which became necrotic, had little or no effect, on
the protein (Darcy, 1957) and the fact that the August D.177 tumour which
becomes considerably necrotic caused only a small increase in titre of the jirot cin
and showed no relationship between the amount of necrosis and the level of t he
protein.

It may be asked whether the level of this protein would be of any use as a
diagnostic tool. For tumours in rats the answer is no. Although the level increascfl
dramatically after implantation of the Walker tumour it increased only r’cry
slowly with the growth of certain other tumours, and tumours of aboiit i-5 cni.
diameter could be present without an elevation of the level above the normal range.
It probably depends on the growth rate of the tumour. As a ])rognostic tool (jio
level of this protein could conceivably have considerable value, esi)ccially in
following the effects of treatment and in detecting relapse.s or metastases. It
would be necessa^, however, to rule out the interference of other influences,
which might be difficult. In any case, the specific protein or an analogous one,
has yet to be identified vdth certainty in human serum. °

StraEttARY

‘“"■O-'S "Itl'ongU

August PtVA.2 tuloOTand tTSe! of •''■o

5. There was evidence that thp nrr,to- ^7 Protein in the blood,
tumour. Tliere is other evidence compatibirw-tl°^ ongmate at the site of the
PC ectively absorbs the protein from the^surrounding fluidl'^''^

546

D. A. DABCY

I sliould like to express my gratitude to Professor Alexander Haddow for
pointing out to me the potentialities of the gel dilFusion technique upon which
this work is based.

This investigation has been supported by grants to the Chester Beatty Eesearch
Institute (Institute of Cancer Research : Royal Cancer Hospital) from the Bledical
Research Council, the British Empire Cancer Campaign, the Jane Coffin Childs
Memorial Eund for Medical Research, the Anna Fuller Fund and the National
Cancer Institute of the National Institutes of Health, U.S. Public Health Sendee.

REFERENCES

Catchpole, H. R. — (1950 Proc. Soc. exp. Biol., N.Y., 75, 221.

Daecy, D. a. — (lQ55)Nahire, Bond., 176, 643. — (1957) Brit. J. Cancer, 11, 137. — (1960)
Ibid., 14, 524.

Dontenvtll, W., Ranz, H. and Mohe, U. — (1959) Munch, med. Wschr., 101, 1365.
Elson, L. a. — (1958) Trans. R. Soc. trop. Med. Hyg., 52, 212.

Korngold, L. and Van Leeuwen, G. — (1957) J. Immunol., 78, 172.

OXYLIC ACID

M. B, SAHASEABUDHE, M. K. NEROTKAR JI. Y. XARURKAR.

B. B. TILAIv AKD M. 1). BHA\ SAR

From the Biology Division, Atomic Energy Establishment Indian Cancer Jicsanch Centre.
Parel, Kmbay 12, and the University Department of Chemical Tcchnnlagy,
Matiinga, Bombay, India

Received for publication July 1, lOGO

Iin a previous communication Sahasrabudhe (1958) put forth cvKlencc in
support of existence of a chemical competition between nucleic acid and p\ ridinc
nucleotide syntheses for the appropriation of a common precursor, adenine. In
a rapidly growing malignant tissue, adenine was shown to be appropriated for
nucleic acid synthesis and very little was left for incorjioration in pyridine nucleo-
tides (Narurkar, Kumta and Sahasrabudhe, 1957). It was further shown that the
pjTidine nucleotide levels were invariably lowered in presence of rapid nucleic
acid synthesis irrespective of whether it (the nucleic acid synthesis) was of neo-
plastic or non-neoplastic origin (Jedeikin, Thomas and Weinhouse, 1950 : Kotnis.
Narurkar and Sahasrabudhe, 1959). PjTidine nucleotides have an important
role in the hydrogen transport system and thus indirectlj- partieijiate in the
production of energy, rda the tricarboxj’'lic acid cj^cle. In view of this it was sug-
gested that low levels of pjTidine nucleotides automaticalh' slow down all the
sjmthetic and proliferative activities bj' controlling the energj" production.
Based on these ideas, a biological feed-back mechanism was postulated for the
regulation of normal growth processes (Sahasrabudhe, 1958). In tumour tissue
however, this feed-back-mechanism seems to break down ; the tumour apparent Ij-
is able to obtain an unlimited supply of energy to maintain its rapid nucleic acid
sjmthesis. A search for an alternate pathwaj^ capable of producing energv
independent of the proposed feed-back-mechanism revealed that hexose-niono-
phosphate pathway (BAIP) has the requisite potentiality of not onlv producing
energj^ (though not yet definitely established) but also jnelding ribose-5-phosphate
which is the starting material for the biosjmthesis of purines. In the hVht of
this the reported preponderance of BBIP pathway in tumour tissue acquires OTeater
significance (Kit, 1956 ; Et and Graham, 1956 ; van Vais, Bosch and Emmelot
19.06) Interference of BCMP pathway, it was thought, would inhibit the tumour

and ribose-5-phosphate. This has been
“c suiLlo intermeSfo"

. Wo., US to out the R-'

548

SAHASRABUDHE, NERURKAB, NABURKAE, TILAK, BHAA^SAB

bolites of glucose-6-phosphate and fractose-6-phosphate (Sahasrabudhe 1958)
These two intermediates are common for both the Embden-Meyerhof (EM) and
HMP pathways, and hence the antimetabolites of these would interfere with the
fflilP and also the EM pathway and thus may prove harmful to the normal host
tissue. Antimetabolites against 6-phosphogluconic acid, sedoheptulose and
erythrose, however, Avere free from this objection. In the early stages of HJIP
patlnvay the carboxyl group of 6-phosphogluconic acid is liberated as carbon
dioxide leaAdng the rest of the molecular arrangement intact in ribose-5-phosphate.
To be effective, the anti-metabolite has to have some functional resemblance to
its normal counterpart ; it is only then that the anti-metabolite can possibly com-
pete for active sites and block appropriate enzyme systems. The anti-metabolite
of 6-phosphogluconic acid therefore should have (i) a free carboxyl group at one
end and (ii) a free or phosphorylated hydroxymethyl group or a carboxjd group at
the other end. Any deliberate departure tlien in the structural configuration of
central (CHOH)4 grouping of the 6-phosphogluconic acid molecule would result
in a substance hardng anti-metabolite properties. These considerations and also
the fact that gluconic acid is readily converted to a 5-membered y-lactones
structure (Fieser and Fieser, 1960) prompted us to suggest that furans, tetrahydro-
furans and also thiophenes and tetrahydrothiopliene derivatives of type (I),
(II), (III) and (IV) might inhibit malignant grou’th through interference of HJIP.
It may not be out of place to mention here that 5-hydroxy methyl-2-furfuralde-
hyde (Heaton and Robinson, 1948) and 5-nitro-2-furfuraldehyde (Friedgood and
Green, 1950) liaAm been showm to have potent cancer inliibiting properties.

A. As

R, O B,

B^ 0 B,
II

B^S Rj
III

B, = B: = H, OH, OEt

R3 = COOH, COOEt, CH„OH, CHo— O— P

,OH

Bj = COOH, COOEt, CH5OH, OH,— O— P

Ribose-o-phosphate is derived in the body not only by the oxidative deea
xylation of 6-phosphogluconio acid ; it can also arise through g.

transketolase and transaldolase enzymes. For effectnm iMnbition of r ^
phosphate formation therefore, it may be necessary simultaneous j , ,

Uh 6-phosphogluconic acid as well as mth erythrose
phosphate. A comprehensive programme of synthesis and testing o c
properties of compounds of the types mentioned is m 9 .

paper records the synthesis and anticancer properties of thiopliene - •

carboxylic acid (TDA).

PROPERTIES OF THlOPHENE-2

: 5-lMCARROXYlilC

AGIO

EXPERIMEXTAIi

Synthms and properlies of thiophcm-2 : o-dicarborylic acid (TDA)

All the four possible thiophene-dicarboxylic acids arc known Vl'.oriP

Sice 19M Snfeld and Jones, 1954). Among tlic difTcrcn methods ava, b o
for synthesis of thiophene-2 ; 5-dicarboxyhc acid the two mo.st coincnient. n] \ .
to be the following : —

i¥ef/iodl.— Thiophene -^2 ; 5 -dichloromethylt!uo])hcnc ->2 : o-diaceto.vymcthy-

thiophene^Thiophene-2 : 5-dicarboxylic acid (TDA). ^

Method 2 .— Thiophene ->2 : 5-diiodothiophcne->2 : .o-dilithmm dci n a1i\
TDA

In the present investigation thiophenc'2 ; o-dicarboxylic acid wa.s ])reparcd
by the second method. Tlie 3nelds by tbe first method were never more thaii 21
per cent or so, whereas bj"^ tlie second method the jdcld was 74 ])er cent . J lie
various stages in synthesis are as follows.

Preparation of 2 ; 5-di-iodothiophene from thiophene

Thiophene {16-8 g.; 0-2 mole) and benzene (20 c.c.) were taken in a glass sloj)-
pered flask, and iodine (lOo g.; 0-42 mole) and yellow mercuric oxide (75 g.; O-.'lo
mole) were alternatelj'- added in small quantities during 2 hours with constant,
shaking and occasional cooling to keep the temperature between 90-45°. 'J'iie
yellow mercuric oxide turned crimson red because of its conversion to mercuric
iodide. Vigorous shaking is required and if the absorption of iodine is inconqiletc
the mixture has to be shaken for an additional hour. The mixture was filtered
and the residue rvashed with benzene and ether. The ether-benzene filtrate was
washed several times irith a 3 per cent solution of sodium thiosulfate till it was
free from traces of iodine. It was then washed with water and dried over calcium
chloride. On removal of the solvent 2, 5-diiodotliiopliene was obtained as a
brownish oil (55 g.). It was distilled at 116-17° /3-5 mm. The small amount of
monoidothiophene (2 g.) present was separated as a low boiling fraction. I’hc
brownish oil (43 g., 64 per cent yield) gave a red-brown solid on cooling. Crystal-
lization from alcohol gave white plates m.p. 40-41° (Minnis, 1943 ; m.p. 40-41°).

Preparation of phenyllithium (Evans and Allen, 1943)

A 250 e c. three-necked flask was fitted with a mercury -sealed stirrer, a dropping
unnel and a reflux condenser. SmaU pieces of litliium (21 g.; 0-3 gram atom)
(prepared by hammering the metal into a thin sheet and cutting it into small
thm strips) and 50 c.c dry ether rvere placed in it. A slow current of nitrogen was

snip's

through a cotton -plugged funnei in a 500 c c flask unre quickly filtered

550

SAHASRABUDHE, NERURICAK, NARURKAR, TILAK, BHAVSAR

Preimration of thiophene-2, 5-dicarboxylic acid (TDA) from 2, 5-diiodothiophene
{Canipaig7ie and Foye, 1948) ‘■

r (stirred and kept under slight nitrogen pressure) a solution

of wj 5-diiodothiophene (10-5 g.; 0*03 mole) in 50 c.c. ether "vvas slowly added (10
minutes) when a Avhite precipitate separated out. Stirring was continued for 10
minutes and the mixture poured into a beaker containing Dry Ice and allowed to
stand for 1-2 hours till aU Dry Ice disappeared. The mixture was then acidified
with ice cold dilute hydrochloric acid (1:1; 10 c.c.) and aUoired to stand over-
night, when most of the ether evaporated olf. The white sohd (4-3 g.) was filtered
and washed 111411 ice-cold w'ater. It was then dissolved in dilute sodium bicar-
bonate, decolorized with Norit in the cold and filtered. TDA was then precipitated
by acidification when a white product (3-5 g.) was obtained. Some more product
(0-5 g.) was obtained from the aqueous filtrate, by extraction with ether. The
ether solution was again extracted with sodium bicarbonate solution and the
purification ivith Norit and precipitating the product by acidification was carried
out as indicated above. The crude acid (4'0 g.; yield 74 per cent) crj^stallised
from water in white needles.

M.P. 322-26° open capillary, slirinking at 318°, effervescence at m.p.

(Found : C 41-8 per cent ; H 2-6, S 18-5 per cent C6H4O4S requires C 4bS ;
H 2-3, S 18-5 per cent).

Derivatives — 2 : S-Dicarbethoxythiophene.

M.P. 50-51° (Friedgood and Green, 1950 ; m.p. 51-51°).

(Found 0 53-5 ; H 6-4 ; S 14-5 CjqOHjj O 4S requires C 52-6 ; H 5-2 ; S 14-0
per cent).

Biological testing of TDA

Anticancer activity of TDA was tested on tw'o transplantable tumours (1)
rapidly growing Yoshida sarcoma (ascites) in Wistar rats and (2) comparativel.v
slow growing solid fibrosarcoma in Sndss mice. Yoshida sarcoma was obtained
through the courtesy of Professor Druckerey (Freiburg). The solid fibrosarcoma
used in the present investigation was initially obtained bj'' Waravdekar and
Ranadive (1957) from animals treated i\dth 6:12 dimethjdbenzo (1 ; 2-b : 4 ; 5-b )
dithionapthene. This has since been maintained in Swiss mice through several
serial transplantations.

Influence on Yoshida sarcoma (ascites)

0-5 ml. suspensions of Yoshida ascites sarcoma cells were injected intraperi-
toneally in 3-4 months old Wistar rats. Thiophene 2 : 5-dicarboxyhc acid
(TDA) was disolved in 0-5 per cent NaHCOg (1 mg./0-5 ml.). Since the substance
is water soluble it was thought that it would be easily excreted out.
injections per day were therefore tried in an attempt to maintain adequate rUo
concentration in the system. Secondly for sustained inhibition of tumour S™''., ’
the concentration of anti-metabolite in the body has to be more than la 0 1
normal counterpart. The dose of anti-metabolite therefore had to be determmeu
depending on the growth rate and the extent of tumour groiHh. n y u o
animals the growth of the Yoshida sarcoma is fairly rapid and results m ® ‘

of all the transplanted animals by about the 4th or 5th day after transp ‘ ’

In older animals the growth of the tumour is known to be comparative y

properties of thiophene-2 ; 5-ihcarhoxylic

ACID

nf) 1

Thp influence of TDA on slow taimour growth was also investigated by transplant-
IX Zrr in 6-7 months oW animnis. TIucc doses mere trie, : ms. once
a day, 1 mg. tivice a day (daily dose 2 mg.) and 1 mg. thrice a day (daili dose ■
Iniections of TDA were evenly spaced during the day ; the corresponding cmitn
aSmTreceiXnly 0.5 ml.'of 'the solvent (O n ,.er cent Nal lOO., solu »„) mtn,.
peritoneaUy. Survival of treated and control animals was noted. Hie results
are given in Table I.

Table I . — Infittencc of TDA on Survival of } oshida Snvenind
(Ascites) Bearing Bats

Treatment and
frequency of
injections

(1) 1 mg. TDA OJJCE a day .

(2) 1 mg. TDA TOTCE a day .

(3) 1 mg. TDA THRICE a day

(4) 1 mg. TDA TincE a day .

.Mean sim'ival time

Total

Ape of

(in

days)

daily do,=o

ral.s

/—

..A

(mg.)

(months)

Control*

TDA treatedt

3-31

r.-o

(i ■ .5

4-2

c.-n

3 !

4-2

fi-a

f.-n

!t-r>

3(MI

* Control group received intraperitoneal injection of solvent (0-5 per cent XallCOj)
t TDA •was dissolved in 0-5 per cent NaHCOj (concentration 2 mg./l ml.) and administered
intraperitoneally.

t The growth of tumour in older animals is comparatively slow.

Influence on solid fibrosarcoma

In the case of solid fibrosarcoma tlie tumour mass was cut into fine pieces with
scissors and a homogeneous suspension ivas prepared in normal saline. O-.") ml.
of the suspension was injected subcutaneouslj' through an 18 gauge needle in
each of the Swiss mice. Since the ceil count of each inoculum could not be taken,
the tumour was allowed to grow in all animals for 7 to 8 days before the treat-
ment was started. On completion of this period animals having comparable
tumour sizes (by \dsual observation) were selected and divided into two groujis.
The experimental group Avas injected Avith TDA dissolved in 0-.5 per cent AhiHCOa,
Avhile the control group received the injections of the soh-ent (0-.7 ])cr cent
NaHC 03 ). In these experiments also the influence of one injection and multijile
injections per day Avas investigated. After 10 to 15 days of treatment the
animals AA^ere killed and the AA'eights of the tumours determined The results are
given in Tables II and III and in Dig. 1 and 2.

Table II. — Influence of one Injection Per Day of TDA on the
Growth of Transplanted Fibrosarcoma in Swiss Mice

Control*

DaiU' dose

0-5 ml. of 0-5%
XaHCOj only

Frequency of
administra-
tion

Onee daily

Day of
starting
treatment after
transplantation
1st

Period of
treatment
(days)

TDA-

trentedf

1 mg. TDA in 0-5
ml. of 0-5%
XaHCOj solut-
tion

1st

AA-ernge weigh I
of tumours nrid
rnnge
_2-Sr.

(I g.)

2- 1 mg.

(1 •.59-2-07 g.)

652

SAHASBABUDHE, NEUVHKAR, ATAEUKKAE, TILAK, BHAVSAE

Table III. — Influence of Multiple Injections per dap of TD A on
the Growth of Transplanted Fibrosarcoma in Sioiss Mice

Time of

starting

Control*

Daily dose

0-5 ml. of 0-5%
XaHCOa only

Frequenej' of
administra-
tion

. Twice dailA-

treatment after
transplantation
(days)

Period of
treatment
(days)

Average weight
of tumours and
range
S-2g.

(O-SO-S-lSg.)

TDA-

treatedf

2 mg. TDA dis-
solved in 0-5%
NnHCOj solu-

. 1 mg. twice
daily

3-16g.

(1- '9-3-90 g.|

tion

j. ""os allowed to grow for 10 days. Then the animals having uniform tumour sizes were

divided into control and experimental groups.

* Tlie control group was injected with corresponding volume of the solvent, i.e. O-o per cent
NuHCO, solution.

t TDA was dissolved in 0-5 per cent IfaHCOj. Everj' time 1 mg. dissolved in 0-5 ml. of O-.i
per cent ICnHCOa wns injected.

In vitro studies on interference of HMP

In addition to the in vivo screening of antitumour activity it was necessan'
to ascertain whetlier TDA really interfered \vith the HJIP pathway. This has
been investigated in in vitro studies by incubating the tumour tissue with glucose-
1-i^C and glucose-6-i‘*C, witli and Anthout the presence of TDA. Six to eight Aveeks
old Saauss mice AA'eighing betAveen 20 and 25 g. AA'ere used. Solid fibrosarcoma Avas
transplanted subcutaneousl 3 L Tumours Avere dissected out tliree Aveeks after
transplantation and Jiomogenised in Potter Elvehjem glass homogeniser in (5
A'olumes of ice cold medium containing 0-133 m phosphate buffer (pH 74). Ali-
quots representing 80 mg. of tumour tissue Avere added to chilled Warburg
vessels. The incubation medium consisted of the follouing substances expressed
in their final concentrations (Wenner and Weinhouse, 1956) : — ^Potassium fu-
marate 7 X lO-^sr ; cj^toclirome C 4 x ; DPN 2 X 10~®M ; phosphate

buffer (pH 7-4) 6-0 X ; MgSO^ 3 X IO-^m ; KCl 14 x lO-bi ; glucose

0-020 jU and gIucose-l-^'‘C or glucose-6-^'^C (as the case may be) equivalent to
6-42 X 10* cpm. 1 mg. of thiophene 2, S-dicarboxj'lic acid dissolved in 0-5 c.c.
of 0-5 jAer cent NaHCOg AA^as added to the medium. A rolled filter paper soaked
in 0-2 ml. 10 N HaOH AA^as placed in the central AA-eU of Warburg flasks to absorb
the CO, released. 0-3 ml. 50 per cent trichloroacetic acid aaas placed in the side
arm. The flasks AA^ere attached to manometers and the assemblj' shaken for 4
hours. At the end of tin’s period, trichloroacetic acid aaas tipped in tlie reaction
A’^essels to stop the reaction and to liberate COj from the incubation medium.
The flasks AATre shaken for an additional period of 60 minutes to allow comp e c
absorption of CO, by the alkali. The manometers AA-ere then detaolied and tJie

EXPLANATION OF PLATE.

Fig. 1. — ^Inhibition of growth of transplantable fibrosarcoma with TDA treatment.^ DosO
2 mg. /day. Treatment started 10 days after transplantation and eontmued lor Jo o.. .•
Left hand— treated with TDA ; right hand-control. ■ i„-|,;f;nn of

Fig. 2.— The same animal as in Fig. 1. With the tumour exposed. :Note the inhibition
growth in the TDA treated animal. Left hand— treated with TDA; right hand-contro .

PROPEETIES op thiophene-2 ; 5-IHCA1UU)XYE1C acip

r.r.ii

(1-5-2 mg./cm-). The results are given in iable IN .

T 4 ULE lY —Iniluence of Thiophene 2, r,-Dkarhox;/lic nr.irl on the Lihnvlion of
JRadlaJve ^^CO/fwm Glncose-W^C G/aco.sc-O-'^C' .Suhstralr. In, I unwur

Tissue RiulinncI ivo '*('0.

Substrate Additions piv.-noiit

Glucose-l-itc . • ■ -J

Glucose-1 UtC . Ti\iopUcnc-2,5.dicnrboxylic acid i t

Glucose-G-*^C . ■ ' .,‘,I

Glucose-6-i^C . ■ Tluophcne-2,;j-dicnrboxyIic acid .

RESULTS AND DISCUSSION

It will he seen from Table I that there was not much difTercnce in the survival
of the control and treated animals when only single injection of TDA (I mg.)
was given everj^ day. Wien the frequency of injections and daily dose was
increased, the TDA treated animals survived longer. When tlie dose and fre-
quency of daily injections were increased and at the same time the growtii of tiie
tumour was slowed down (as in old animals) the results were striking. In Mii.s
group the treated animals survived up to 30 days whereas the control animals
died by the 12th day after transplantation.

Similar results were obtained u-ith the solid fibrosarcoma in Swis.s mice.
MTien the daily dose was 1 mg. once onlj’^ the weights of the tumours in tlie control
untreated animals were not markedly different from those of the treated animals.
But when the frequency and the daily dose was increased, significant inhiliition
of tumour growth was eindent in the TDA treated animals. Thus whereas the
mean weight of the tumour in the control animals was 8-0 g. that of the treated
groups was 3-0 g. This is clearly seen in the photographs of the treated and
untreated animals (Fig. 1 and 2).

In the in vitro studies with glucose-l-^^C and gluco.se-6-i^O it was seen that
liberation of radioactive i^C02 from glucose-l-”C was diminished in presence of
TDA suggesting that the HMF was probably inlribited by the presence of TDA.
It will be noticed that in the presence of TDA the liberation of radioactive * 'C0„
from glucose-6-i^C substrate was also diminished. This is due to the fact that in
tumour tissue a portion of trioses is obtained via the EOIP pathway. If this is
so then the diminution m the evolution of radioactive CO, when glucose-6- J >C
,s to be expeeted. In vitro stato thus seem to jusfify the pr”m sc thet

imlpf i'SmtdLr If S ‘'-ose-mono.phosphate

(TDA) h.s been sjmthesled Id 57,^1 ^ ^ 5-dicatbo.vylic acid

i-st(g..ed on YosMda (ascites, satcoma inlS'Inn^nfl',: si"

554

SAHASRABUDHE, NERUBICAR, NARUEKAE, TILAK, BHAVSAR

fibrosarcoma in S^viss mice. The smnfival of Yosliida sarcoma bearing rats was
increased AWth TDA treatment, whereas Avith the solid fibrosarcoma a significant
inhibition of tumour growth Avas evident. In vitro studies carried out with
gIucose-l-i''C and gIucose-6-i^C suggest that TDA interferes Avith the HilP
patliAAmj'’.

REFERENCES

Cajmpaigne, E. and Foye, W. 0.— (1948) J. Amer. chem. Soc., 70, 3941.

Ea'ANS, J. C. W. and Alden, C. F. M. — (1943) Org. Synth., Vol. II, p. 517.

Fieseb, L. and Fieser, M. — (1960) ‘ Organic Chemistrj^ ’. NeAv York (Reinhold
Publishing Corporation), p. 377.

Friedgood, C. F. and Green, M. N. — (1950) Cancer Res., 10, 613.

Hartough, H. D. — (1952) ' Thiophene and its Derivatives New York (Interscience
Publishers Inc.), p. 402.

Heaton, T. B. and Robenson, G. M. (1948) Aa/nre, 162, 570.

Jededon, L., Thomas, A. J. and Weinhouse, S. — (1956) Cancer Res., 16, 867.

Kjt, S.— (1956) Ibid., 16, 70.

Idem AND Graham, 0. L. — (1956) Ibid., 16, 117.

Kornfeld, E. C. and Jones, R. G. — (1954) J. org. Chem., 19, 1671.

Kotnis, L. B,, Narurkar, M. V. and Sahasrabudhe, M. B. — (1959) J. sci. industr.
Res., 18C, 63.

AIinnis, W. — (1943) Org. Synth., Vol. 11, p. 357.

Narurkar, M. V., Kumta, U. S. and Sahasrabudhe, M. B. — (1957) Brit. J. Cancer,
11, 482.

Sahasrabudhe, M. B. — (1958) Nahtre, 182, 163.

Sice, Jean (1954) J. org. Chem., 19, 70.

VAN Vads, G. H., Bosch, L. and Ejimelot, P. — (1956) J. biol. Chem., 222, 399.
Waeavdekae, S. S. and Ranadive, K. J.— (1957) J. nat. Cancer Inst., 18, 555.
Wennee, C. E. and Weinhouse, S. — (1956) J. biol. Chem., 219, 691.

®PLKi?S)N TO THE USE OF OXYGEN IN BADIOTllElIAn

R. H. THOMLIKSON

From the Medical Besearch Council, Experiment^ HadinpM^^^^^^ Unit.
Hammersmith Hospital, Ducatic Road, London, 11 .1-

Received for publicntion July 1, 1000

When the results of radiobiological experiments (Gray, Conger, Lbert,
Hornsey and Scott, 1953) first led to the use of oxygen with radiotIicrai>y for human
cancer (Churcliill-Hayidson, Sanger and Thomlinson, 1955) the need for a satis-
factory experimental system for comparing differing schemes of treatment, became
apparent. A system using malignant tumours in the rat for com])aring t he results
of many forms of cancer therapy has been developed. Tins will be described to-
gether with the results of the first of a series of experiments using radiotheraji.v
and different conditions of oxygenation.

The Principles of an Experimental Si/stcm

It cannot be over emphasised that in using a biological experimental system,
the variables betAveen one test and another should be the relatively simple and
measurable physical and chemical factors, whilst the effect on the complex and
usually immeasurable biological factors should be made to match. In the context
of this investigation the question is being asked “ to what extent does the con-
centration of oxygen reaching a neoplasm modify the dose of radiation required
to produce a particular result? ”

Answers to this question can never be obtained from human radiotherapj^ since
one patient varies so much from another and no tAVO neoplasms are exactly alike.
Moreover there are narroAv ethical limitations to the variation of treatment and
in general only one result may be sought. If hoAvever, the experimental testing
of therapy in animals is to influence clinical practice, the pathological principles
underlying the experimental system and the human situation must be understood
and be seen to be identical ; the more nearly the experimental conditions simulate
those of the treatment of human cancer the better.

Tor these reasons it has seemed essential to use an animal neoplasm AAdiich

“f f re,ZaKo'"4“1

a randomising process must be introduced to determinTfl”^?

g,van cacK inaUaea and the reaulta he Bj«S%o:taSera‘„ab.£

556

R. H. THOMLINSON

The Technique of Transplaniation used for Producing Tumours for Expmmtnl

, Tlie difficulty of using a malignant neoplasm is that metastases niav fnm,
before the treatment is given and lead to the death of the animal before the effe
of ti>eatment on the primary tumour can be assessed. After many failures to
obtain results fOT this reason a somewhat elaborate technique which appears to
overcome tJie difficulty has been evolved. “

Rats have been used for these experiments because of their convenient size
® strain derived from a single pair of Wistar siblings in
193J. llie tumour used so far is a fibrosarcoma induced in the strain bv sub-
cutaneous injection of benzopyrene in 1945 and knovm as BIBS.

The tumour has been maintained by subcutaneous grafts in the flank. From
such a graft, healthy^ tumour tissue is minced until scissors and mixed with a
solution of sodium alginate in the approximate proportions of two parts of tumour
to one of alginate. This mixture is then dropped from a fine hjqiodermio needle
into a 1 per cent solution of calcium chloride. A gel of calcium alginate is formed
so making a capsule to the drop, the thickness of which increases m’th time. As
the technique is now used, 20-30 seconds is sufficient to produce a tumour
pellet which can be handled in a pipette and transferred after this time to a
physiological saline solution.

Meanwhile a “ sausage skin ” has been prepared bj'^ taking the small intestine of a
freshly killed tliree week old rat, inverting a length of it and wiping off the mucosa
with a gauze swab. Tumour “ pellets ” are now placed within the lumen of the
inverted intestine which is tied on either side of each pellet and cut into sections
to produce almost spherical tumour “ sausages ” each about 3 mm. in diameter.

A skin incision just large enough to admit a pipette containing tlie tumour
sausage is made in the abdomen, and then a burrow in the subcutaneous tissue
layer immediately deep to the panniculus carnosus extending as far as the position
in the flank where the tumour is to grow. A tumour sausage is then put into the
blind-end of the burrow uith a pipette and the skin incision closed.

Most of the tumours transplanted in this waj^ gi'ow to form rounded masses,
in the clinical sense unattached to skin or muscle, reaching a diameter of 10 mm.
in about fifteen days (Fig. 1).

EXPLANATION OF PLATES.

Fio. 1. — ^A rat -with a solitary, localised, smooth and rounded subcutaneous tumour in tho flank.

This is a random example 9 mm. in diameter.

Fig. 3. — ^Photograph of pressure chamber with upper hemisphere removed. The gas mter-
tube and needle valve are in the foreground. Beneath the chamber is the box containing
the heating coil and behind it, the cooling columns. Two lend colimater rings are on the bo.x
and one is in place abov-e the couch which supports the rat. On the right is the box
containing the eleetricallj^-operated valves controlling gas flow and in front of this he.? tho
“ U ’’-shaped clamp for making the tumours anoxic.

Fio. 14. — Photograph of part of the cut surface of a fixed tumour RIB5 about 40 my. in
diameter. The edge of the tumour is on the left. Near this is a zone of homogeneous infnc
tumour. On the right all the tissue is necrotic. In the intermediate zone are man}
prominent, dilated and congested blood vessels.

Fig. 15. Photomicrograph of tissue of tumour B.IB5 from the intermediate zone siionn in
Fig. 16. IVhat was homogeneous tissue has broken up into cylindrical systems of nbou
100 /i radius, with a dilated blood vessel at the centre. These blood vessels have wfl is
endothelial cells and basement membranes only. Surrounding them is a zone of m no
tumour tissue. Outside this is a zone of necrotic tissue in which cells are clearly
able and in which the necrosis is relatively recent. Beyond this all the tissue is necto
and details are lost. This necrosis is older.

TJioiiiliiisoii.

JtETHOD TOR COMPARING TOBATMEXTS OP TUMOURS

;>;> /

The Technique for Irradiation Tumours in Differing Pressures of Oxiigeu

All irradiations were made with a Marconi Industrial Model X-rny set, with
an electrical^ operated shutter, running at 250 kv. and 10 or 15 in. a. to give a
dose rate of 400 rads per minute using filters of O-o inm. copper and 1-0 mm.
aluminium. The mid-point of the tumour was approximately 21 cm. from the
target of the X-ray tube. The dose given at each irradiation was controlled by
using a monitor placed in front of the shutter. This consisted of an ionisation
chamber through which the X-rays passed and the current of which was integrated.
This current was coiTelated with the dose received at the jiosition of the mid-
point of the tumour as measured with a condenser-ionisation chamber. Ail the
animals were treated within a spherical pressure chamber 0 inches in diameter

X- RAY TUBE

Fig. 2

LEAD RUBBER SCREENING
LEAD COLLIMATOR RING
TUMOUR LYING BENEATH COLLIMATOR
RAT

COUCH SUPPORTING RAT

AOJUSTA8U WOODEN TABLE
REGULATING DISTANCE O'

TUMOUR FROM SUPPORT RING

thermocouple

COOUNG COIL-

IONISATION eWAMBEP
RING OF MONITOR

dome OF PRESSURE CHAMBER

Fitting against monitor

'SUPPORT RING )N CHAMBER A
FIXED DISTANCE FROM DOME

SPIDER PLATFORM SUPPORTED
ON A FIXED RIH

PRESSURE gauge

HEATlVC cOfL

showing th?mechanisms^fo7comroilw'’g;ls^o ’"'s'" Pressures of o.vvpen

and protecting the rat and the relatiol^of the oWbeT'?rnrSonao"'^nnd'kv^^

vvere bolM

the f'C ± C. hy passing

water cooled spiral. These alternative chaS.^ ^ eJectricallr- or through a

gas pressure was regulated hr?! Brittr n minutes Tim

rosulntcd bv . n i e vSv, f ''S”' <>" -haraW T,?'"''"-

htyoml this'. “ ‘It' «-t from the chemiTitith e J'l

The anaesthetised rat was ^ ^ flo"'-nieter

558

R. H. THOMLINSON

Tliis coucli was attached to a wooden piatform carried on a Perspex “ snidpr ”
resting in turn in the lower liemispliere of the chamber. Also carried bv this
spider , immediately above the rat, was a horizontal metal ring about the
^mrtical axis of the chamber. In this ring could be placed one of several rings of
lead, one inch in thickness, used to define the X-ray beam. This lead rinAvas
therefore in a fixed position in relation to the lower hemisphere of the chamber
and hence the upper hemisphere when that was in place. The upper hemisphere
was brought into relation with the monitor on the outlet of the X-mj tube. Tlie
distance that the tumour lay from the X-ray tube was adjusted by a vertical
movement of the couch and Avooden platform in relation to the lead defining ring.
A single measurement from the mid-point of the tumour to the under surface of
the defining ring ivas all tliat needed to be made in calculating the monitor
reading Avhich indicated the selected dose.

It was possible by palpation to define the position of the deep surface of the
tumour and the adjacent abdominal muscle and this rvas marked on the skin.
With the animal Ijdng on its side, the centre of the tumour Avas placed in the axis
of the chamber in such a Avay that a Amrticle beam of X-rays Avas tangential to the
abdominal musculature. A suitabl 5 '' sized defining ring was chosen to ensure
irradiation of the AA’hole tumour and protection of the intestine. The rest of the
animal AA-as coAmred b}^ a sheet of lead rubber.

Procedure

The rats AA’ere immobilised b}’' anaesthesia produced AAuth an intra-peritoneal
injection of sodium amylobarbitone, 12-5 mg. for a 200 g. rat, and proportionately
slightly less or slightlj" more Avitli lighter or heaAder animals.

Wlien irradiation Avas to be given Avith the tumour anoxic a clamp made of
tAvo horse-shoe shaped pieces of Perspex AA'as placed on the skin around the tumour,
betAA'een it and tlie abdominal muscle and tightened to occlude the circulation.
The animal Avas placed on the couch in the pressure chamber, Avhich aa’ss then
closed and air passed through it at a rate of about 1 litre per minute. The clamp
Avas left in position for six minutes before radiation began and removed im-
mediately after its completion.

For irradiation to be giA^en A\dth the animal breathing air sodium anijdobar-
bitone aa'us giAmn in the same way and the rat placed in the pressure chamber AA’ith
no interference to the tumour circulation. The chamber Avas closed and air
passed through it. Irradiation AA^as gh'en iramediatety. On some occasions,
after completion of irradiation, oxygen aa^s given at pressure in the manner used
for irradiation in oxygen.

When irradiation Avas given AAuth the animal breathing oxygen at Ingl) pressure
it AA'as anaesthetised and placed in the chamber in the usual Avaj'. Oxygen aass
passed through the chamber at 10 l./min. to flush out the nitrogen. The pressure
Avithin the chamber Avas then raised at a rate of 10 lb. /min. The earliest rise m
pressure sometimes caused moAmment of the animal. This could be stopper or
minimised by raising the pressure more sloAAdy. The extent of any moA'cme
could be seen from the marks on the skin, and if need be the chamber aa as open
and the process started again. The pressure of oxygen was raised to 45 lo. pe
square inch (4 atmospheres absolute) and maintained for fifteen mmu es
irradiation.

METHOD TOR COMPARING TRPIATMENTS OP TUMOURS

a.');}

Immediately before and immediately after irradiation in cacli condition Mu-
position of the tumour was inspected and the respiration rale measured. During
treatment the animal was observed visually and the thermometer, pressure gang

and sas flow meter could be seen. i i i

After the completion of irradiation in oxygen the pressure was lowered siowlj .

After using pressures of 30 lb. per square inch five minutes appeared to lie a long
enouc^h time, but after 45 lb. per square inch pressure thirty to forty minutes were
required, if damage to lungs or nervous system were to be avoided.

The Design of the Experiment

This experiment was designed to test the hypotheses that in an intact, malignant
tumour there are cells which are not fully oxygenated, that tlicse, after aerobic
irradiation, are capable of regenerating the tumour in its onviroiiment and that
they can be influenced by the breathing of higher ])rcssures of oxygen.

The effect of irradiating tumours was therefore tested at two doses, 2000 rads
and 4000 rads, and under each of three conditions, (i) the tumour being made
anoxic by stopping its circulation, (ii) the tumour “ aerated ” with the rat brcatli-
ing air, and (iii) the tumour “ oxygenated ” by the rat breathing oxygen at 4.5 lb.
per square inch pressure. Results were obtained by making daily measurements
of the diameter of the tumour in each of the three dimensions with graduated
calipers and taking the arithmetic mean of these three measurements.

In addition to these irradiation “ treatments ” six types of control “ treat-
ment ” have been carried out. In the first, onlj-^ daily measurements were made.
In the second, no irradiation was given but the animals were given the anaesthetic.
In the third, the anaesthetic was given and the circulation to the tHmoiir.s was
occluded for twenty minutes. In the fourth the anaesthetic was given and the
animal placed in oxygen at 45 lb. per square inch pressure for twenty minutes.
The fifth type of control treatment was surgical excision of the tumour'performed
to test the frequency of metastasis formation before the time of treatment. The
sixth control treatment consisted of giving the rats oxygen at 45 lb. per square
inch pressure for 20 minutes after irradiation in air or after irradiation of the
tumour in the anoxic condition.

Measurement of the growing tumour could be made from diameters of about
o mm. upward. ^1 treatments were given when the tumours had attained a
mean diameter of between 8 mm. and 10 mm. Each form of treatment was given
a number and was aUotted to the tumours from a table of random numbers as the -
reached this size. The pre-requisite conditions for any treatment to be given were
that the tumour was solitary, smooth, rounded and mobile in the sulfciitaneous
tissue and climealiy not attached to skin or muscle suOciitaneous

these two animals were lost by deat1[from haem of 82 tumours. Of

after decompression in oxygen when this immediately

animal having a doubtful addirionarmtute ^

was discarded wlien this grew rrithiTa W of radiation

...scar., .a fro„.

560

R. H. THOMLINSON

Table I

Tj'pe of treatment

Untreated control .......

Control given amylobarbitono onlj' .....

Control given amylobarbitone and clamping of circulation .

Control given amylobarbitone and oxygen at 45 lb. per sq.
in. pressure

Surgical excision ........

2000 rads given to anoxic tumours .....

2000 rads ivitb rat breathing air at atmospheric pressure

2000 rads with rat breathing oxygen at 45 Ib./sq. in. .

4000 rads with tumour anoxic ......

4000 rads with rat breathing air at atmospheric pressure

4000 rads with rat breathing oxygen at 45 Ib./sq. in. .

Of tlie remaining seventy-eight tumours, the distribution amongst the different
forms of treatment and the mean diameter of eacli group at the time of treatment
is shown in Table I.

It should be noted that whilst the numbers in some of the control groups are
still small these can be added to in subsequent experiments. The larger number
of those treated with 4000 rads given in oxygen is due to an additional six being
treated in succession at the end of the experiment.

Result of surgery

In all 1 1 tumours were excised. In one animal there was local recurrence at
the site of the operation. On post-mortem examination no metastases were found.
One of the remaining ten animals died on the 60th da}'^ after excision. In tliis
animal there was no sign of neoplasm at the site of operation or in the axilJarj',
inguinal or iliac l 5 TOph nodes. HoAvever, there Avere massiAm metastases in the
lungs, in the upper mediastinum and around the loAver part of the pericardium
and upper surface of the diaphragm. The remaining nine animals are aa'cH and
AA'ithout sign of desease after more tlian 120 daj'^s from the time of excision.

Results of control and radiation treatments

In this first experiment it has seemed Avorth presenting the curAms relating
the mean diameter of each tumour to the day before or after treatment (R day)
to shoAV the extent of the variation. These curves are shoAAm in Pig. 4 to 11.

In the untreated control group (Fig. 4) all tumours grcAv at a nearly uniform
rate. Of the six animals shoAvn, tAAm died probably from haemorrhage into the
tumour, before a diameter of 50 mm. AA^as reached. In the second control group
(Pig. 6) the groAAdh rate of all tumours, AAdiether treated AAuth anaesthetic only,
anaesthetic and clamp or anaesthetic and oxygen, Avas similar and fell Avithin the
range of the untreated controls. After 2000 rads given AAdth the tumour made
anoxic (Fig. 6) the groAvth rates Avere similar and slightly but appreciably delayed
as compared Avith the control. After 2000 rads given AA'hilst the animal was
breathing air AAuth the tumour circulation unimpaired (Fig. 7) there Avas consider-
able delay in growth and more variation from tumour to tumour. This variation
Avas more prominent after 2000 rads given AAuth the rat breathing oxygen a
45 lb /square inch pressure (Pig. 8). One tumour of this group became impalP'

Uumber of
animals
8
3
3
3

Mean diameter
of tumours
(mm.)

9-4

8'8

8-6

8- G

9'1

9 - 5
9-1
9-0

8 - 5

9 - 0
8-8

JIETHOD FOR COMPARING TREATMENTS OF TUMOURS fidl

able after 29 days and tlic animal is well after 120 days. At the oilier ex'treme
the gro^rth rate of one tumour was similar to that of the anoxic grouji.

After 4000 rads given to the anoxic tumour (Fig. 9) there was once more less
variation in response as compared with tlie air and oxygen groujis and an ajipreci-
able slowing of growth as compared with 2000 rads given to the anoxic tumour.
4000 rads given with the rat breathing air (Fig. 10) resulted in some variaf ion of

Tumour RIB5, Uiiti-catcd coiiti-oC.

response

Biuup treated wi

,, 1‘o^sMe rcasorSl " S oftS'''

reasonable, those in

562

B. H. THOJILINSON

each group whicli follow a common trend (Fig. 13). It will be noted liow the
variation within each group increases from the anoxic group through the air
group to the oxygen group and is more marked with the higher dose than the lower.
The mean survival time of all animals in each group is shovm in Table II.
Not all tumours grew after irradiation to reach the large size of 50 nun. in
diameter. Three tumours Avere apparently cured. Many other animals died of

Tumour RIB6. ITiilrradiatied coiiti'oL.

Days. ( R = tiNotment dt^.)

Fig. 5

Table II

Treatment

All controls . . . ■

2000 rads, tumour anoxic
2000 rads, rat breathing air .
2000 rads, rat breathing o.xygen
4000 rads, tumour anoxic
4000 rods, rat breathing air .
4000 rads, rat breathing oxygen

Jlean survival
time in days

n±i-i

2I±I-6

28±l-3

39±8-2

27±l-7

37±0-8

45±7-l

Mean diameter C nxni.)

method for

COMPARING TREATMENTS OE TCMOCRS

r.n:i

Turaour 1\I55. 2000 rads u’itit tumour anoxic.

D<^s C 'treatment day.^

Fig. C

metastases before the tumours had grown large. However, in eacli group several
tumours have grovm to 50 mm. in diameter, and the average times tliey liavc taken
to do this are listed in Table III.

Table III

Treatment

Controls

2000 rads, tumour anoxic
2000 rads, rat breathing air
2000 rads, rat breathing oxygen
■4000 rad.s, tumour anoxic
4000 rads, rat breathing air
4000 rads, rat breathing oxygeA

Time after treatment
day for growth to
50 mm. diameter
in days
14

(29 after
implantation)

564

R. H. THOMLINSON

No significant result has appeared from
these will not he presented in detail.

analysis of postmortem findings and

DISCUSSION

Tlie object of the investigations of ivhich this paper is a report is the eluciclatinn
of certani problems to do with the use of oxygen in combination with Jadh,

Tumour RIBS. 2000 rods. Rat 6reatftiug air.

1 \ 1 1 1 ! 1 \ L— L-1..I I- i. .1 ■ I _l _ >■ 1 l_ ^^L_L _L I I t_l_ t I I . .1 J

~Z R 2 4r 6 8 10 12 14 16 18 20 22 24- 26 28 30

D(^s ( R * ti*eatmcnt day.)

Fig. 7

therapy in the treatment of human cancer. The first answer to be sought is a
clear demonstration of the beneficial effect of oxygen with radiation in the treat-
ment of a tumour irradiated and followed in situ in the host animal irith no
further interference than the making of daily measurements. Other practica
questions such as the optimal pressure of oxygen which should be breathed, the
minimal time for which oxygen should be given before treatment, the whole com-
plex issue of the fractionation of the total dose, and the dose itself in relation to
tumour size and type are awaiting answers. Clearly these will only come m le

JIETHOD FOR COMPARTXG TUEATMKXT.S OF TUMOFUS

ndf)

light of understanding of the radiobiology of nco])lastic cells, tlio patliology of
tumour growth and the physiology of oxygen trnns])ort. in the animal body and
■within the tumour itself.

Tumour RIBS. 2000 rads. Rat 6i'catfi'uu]
oxygen at 45 C6s.“pi'cssui'e.

Days (R-timtmcrit

Fig. 8

The pathological basis of the experiments
Radiobiological aspects of the effect r^r

of Hio cells. sSnt ’T7 by tlam.Be to tL ' , ™tlteMon and

566

R. H. THOMLINSON

tumour (Desclmer and Gray, 1959) has been assumed, but must be confirmed in
due course.

Direct evidence of the presence of cells at low concentrations of oxj'gen in
intact tumours at the time of irradiation is scanty and uncertain. The probable
existence of oxygen gradients in certain types of carcinoma such as would be
likely to cause low levels of oxygen availability to some cells has been suggested

Tumour RIB5, ^000 i*ads wltfi tumour anoxic.

Dc^s (K-ti'catmentdc^.)

Fig. 9

(Thomlinson and Gray, 1955). The type necrosis in other

Ust exist was considered in relatmn to the ^^^^iSon (1957).
types of tumour by Churchill-Davidson „ ,vith which a common

^^The importance of this relationship lies in the q ^ of malignant

pattern of vascular disturbance and necrosis s humour RIBS has grown to

growth including human cancer. Dor j j centre has undergone

a large size, say 30 mm. or more of intact tumour

coagSative necrosis and at the peripherj' is an irregu

METHOD EOR COMl'AUIXG TUKATMEXT.S Ol' TOMOn^S

;■)(’) 7

tissue ranging from o nini. to 10 mm. in thicknc.ss. In tlu* oilier jmrt of this zoiu'
there is no necrosis. A little towards the centre, small areas of necrosis apjiear
at points at the greatest distance from blood vessels. In the region bordering on
the necrotic centre the intact cells appear to break nji info cylindrical systems
surrounding dilated and congested blood vessels, t he walls of which are of capillary
structure (Fig. 14 and lo). At the ])cri])hery of these systems are zones of neerot ic

Tumour RIB5. 4000 rads. Rat trcatfiing air.

D^s. ( R - ti'eatmeiit di^.)

Fig. 10

568

K. H. THOMLINSON

sucli capillaries. It seems reasonable to suppose that living cells in these tumours
Avhich are in low concentrations of oxj^gen, if they exist, are to be found at the
greatest distances from such capillaries, that is to say, on the borders of necrotic
areas.

Tumour R1B5. 4000 rads. Kot Greatfiing
O2070cn at 46 Cbs? pressure.

50
45
40
? 35
30

.1 ^
C 20

/■

/:/

rf'

/ y

'f//

-2 R 2 4 6 8 10 12 14 ^ 16

Days (R^ treatment day.)

Fig. II

If this picture of the presence and

right, it seems that in the natural course o radiotiierapist since they

and it may be supposed t^^J ^"? X^an) However, it is a h3Totliesis
cannot become “ more dead than de ( )• surviAm because of the

that after irradiation in aerobic conditions these c ^^^^ Y nutritional

protectiAm effect of hyrpoxia and regener radiosensitiA-e

conditions Avhich follow the death and absorption ot their mo

neighbours.

METHOD FOR COMrARINO TREATMENTS OF TEMOERS

nr.!*

John’s hjqwthcsis (sec Churchill-Daviclson. Sanger and 'I'homlinson, I!)"t7)
suggests the massive necrosis in tumours is due (o venous infarelion following
slowly progressive venous obstruction consequent upon the expansion of t lie tninonr
mass in a limited space. If this were correct it would imply that more oxvgen

Irradiation of tuinoiir RI.65. I .

riG. 12 .

Cflri orilv rGcidi t * •

570

B. H. THOMLmsOJS-

MltoVrlnd '’^'

Irradiation of tumour RIB5. IT.

g o5

t- 50
■§

I ■=

i "

i:^ 16

"^5

/ 2000 nods 4000 mds ,2000fads

•_ ■» . ar I

Gsntrot ■,, ,,

Y ^ y — -jvwyjuo ! i.

j/J Ano-vic Anoxic / / i

r r

// - iL-l

/ /

//
//

in am

/ / K /f
1/ ^

[/^

r/1

4000 rads
_ in ain

2000 rads
in oxwn
0145165°

" 4000 rods
in ox^igon
Qt 46155°

-I I L.

_l I I 1_

' ' ' ' ' ' ' ' ' 1 1 1 1 — I — I — I 1 1 1 [ I I I I I I J I I

-Z R 2 4- 6 8 10 12 14^ 16 18 20 22 24 26 28 50

JD^s. (R= treatment day.)

Fig. 1 3. TJie arithmetic means of the growth curves of those tumours in each group following
a common trend. The standard errors of the means are sho\\Ti. Note the match between tlie
curves after 4000 rads given to the anoxic tumour and 2000 rads given in air and those of
4000 rads given in air and 2000 rads in oxygen.

tumours lie in the realms of tumour immunolog 3 ^ It may well be that no sucli
tumour is isogenic with its host (Prehn and Main, 1957). However, the influence
of immunity can be minimised using in-bred strains of animal and tumours
arising spontaneously or by induction within the strain. The degree of incom-
patibility can be tested by using the cell dilution techniques of Hewitt (1958) and
the four tests suggested by Scott (1960) are being carried out. The effect of
immunological incompatibility between tumour and host is to produce cures
after irradiation when cells have survived the radiation injmy- in such numbers as

METHOP FOR COMPARING TREATMENTS OF 'J’UMOURS *> ' i

would have regenerated tlie tumour if no iniinune reaction existed. The relations
between radiation damage and immunological response are very eomplex and have
recently been discussed (Scott, I960). These inter-relations make the c.xacl.
comparison of the groups in these exjjerinients rather difficult l)ut tlie ])ossible
effects of immunity are diminished when comparison is made between dose and
oxygen concentration which produce equal damage to the tumour.

Transplantation technique

The development of a technique which would admit this tyjic of comjiarison
has proved surjirisingly difficult. At first, small masses of ajiparently^ health}^
solid tumour were implanted subcutaneously, but these resulted in irregular

• CapiOanct Kfccnt nccro«l«

CD Intact tuitiour I OUcr nccroils

I ■ » = 1 mta.

Fig. 16. — A diagrammatic map of tissue in the intermediate zone ns shown in Fig. 14 and 1.6.
The uniform width of the zone of ‘‘ recent necrosis " suggests a dynamic system witli
gradually failing circulation in the “ capillaries ”.

shaped tumours which frequently grew into skin or muscle. Centrifuged free cell
suspensions were then injected into subcutaneous tissue through a fine hypo-
dermic needle, but these tumour cells followed the needle track and the incidence
of early lymph node metastases was high.

At this stage a mixture of a centrifuged cell suspension and sodium alginate
was first used in proportions of two parts to one. This mixture was dropped into
Tt 1 solution of calcium chloride and left in it for varing lengths of time,

lad been determined that the tumour cells alone would grow after ten minutes
solution. It was found that after 20 seconds immersion the
causnlT ^ formed at the periphery of a drop of the mixture produced a
in the subem^* be handled by pipette and be implanted

to form '''bilst almost all these “pellets ” grew
Tile imnrp followed the needle track and infiltrated the ^in

ion. At the other extreme pellets left in calcium chloride for five

572

R. H. THOMLINSON

minutes were almost solid and failed to grow at all. Eventually a time of li
nimutes was selected as giving a reasonable yield of tumours (about 75 per cent
of those implanted).

Irradiation experiments carried out with those tumours resulted in a disap-
pomtmg loss of usable material within a few days following treatment either from
the death of animals due to metastases or the rupture of tumours through the
skin. ®

An extensive series of surgical excisions of the primarj'^ tumours at varying
sizes from 3 mrn. to 12 mm. in diameter resulted in 50 per cent cures irrespective
of size. This indicated that metastasis formation, or spread of the tumour
bejmnd the field of excision and therefore of irradiation, had occurred in the other
50 per cent of cases as a result of the transplantation technique. Experiments
starting from this situation seemed fruitless.

During the course of these experiments it was reahsed that those tumours
which, at the size of 10 mm. diameter, felt smooth and rounded, and which were
in the clinical sense mobile in the subcutaneous tissue and unattached to skin or
muscle, went on to grow to a large size without obvious metastases before a late
stage in the disease. This seemed to be the condition at the 10 mm. size in which
the tumours were suitable for experiment. The problem appeared to be to hold
the transplanted cells togetlier in a mass until the trauma produced by the trans-
plantation process had healed, or at any rate until a barrier had formed around
the transplant.

Recollecting, from the days of keeping pigs during the war, the way of making
sausage skins from small intestine, it was decided to try to make viable tumour
“ sausages ”. In the first place the viability of tumour cells vdthin a capsule of
intestinal wall Avas tested by scraping off the mucosa of the adult rat jejunum and
filling the lumen with minced tumour. This was then tied into suitable lengths
and implanted. The tumours all grew and histological examination showed the
presence, not only of Auable cells, but of ncAvly formed blood vessels both within
and without the remnants of the intestinal Avail. In spite of Avashing and searing
it is quite possible that many Auable tumour cells AA'ere left beyond the “ ties ” at
the end of each sausage, and therefore not surprising that the resultant tumours
AA'ere of irregular shape. The next step, hoAvever, yielded satisfactory results. This
Avas to implant fragile alginate tumour pellets into the lumen of the inA’^erted
intestine of a young rat. The pellets held the tumour cells together until the ties
had been made. These spherical “ sausages ” haA'e given a good yield of tumours
meeting the conditions required for experiment. A fcAV haA'e infiltrated muscle
but this Avas probably due to implantation in too deep a subcutaneous la3'er. The
results of the small series of surgical excisions performed so far indicate that at
the 8 mm. to 10 mm. size the chances of the tumour being still localised are high.

It seems likely that this technique can be used for the implantation of other t 3 'pes
of tumour and aaIII enable in vivo comparisons to be made of other t3q3es o cancer
therapy as aa'cH as radiation.

Irraduation technique

Little comment need be made on the radiation technique except to e/nphasise
the necessit3' of aA'oiding any manipulations AAdrich ma3' impair le
in the tumour, bearing in mind the fragile structure and the
pressures of the veins. Any question of interference Avith these mAahdates th

JIETHOB FOR COMPARING TREATMENTS OF 'I’UMOUHS

573

results of experiments involving oxygenation. Variations of skin circulation
with environmental temperature should also he borne in mind.

The need to reduce the pressure in the chamber very slowly after irradiation
in oxygen became apparent when two animals develo]icd sjiastie ]iara])logia of
the upper limbs. This was probably caused by some embolic iihenomenon in
the spinal cord and although there has been recovery, it is not com])lcte.

The apparatus used in these experiments is being modified to demonstrate
radiographically the presence of the whole tumour in the radiation field im-
mediately before and after the do.se is given.

Results

The most disturbing feature in the results presented from this first ex])crimont
is the scatter of the growth curves following irradiation in air and more still in
oxygen. This is more obvious with the higher dose and most jjronounced in
the separation into two populations seen after 4000 rads, given in oxygen. Al-
though this variability is explicable in terms of small dilTerences in the number of
cells surviving radiation the different re-sponse may come to roHect the difference
between cure and failure in treatment. Five possible explanations may be
advanced.

First, that the tumours growing more rapidly after irradiation in fact contained
a rather larger number of viable cells at the time of irradiation. Provided that
all the cells are well oxygenated, where two out of fourteen tumours have been
completely killed by 4.000 rads, all would be expected to be killed by 5000 rads.
The effect of the small possible difference in numbers at the time of irradiation
would disappear in this case, and the result of experiment with a higher dose will
decide this explanation.

Second, that the whole tumour was not irradiated. Whilst this seems un-
likely, steps are being taken to demonstrate radiographically that the whole of
each tumour is in the radiation field at the beginning and end of treatmont.

Third, that parts of the tumour remained hypoxic in spite of oxygen administra-
tion. This seems the most likely explanation and quantitative predictions of the
effect of the presence of a few anoxic cells are consistent with these results.
(He\yitt, 1959). The use of different pressures of oxygen may resolve this. It is
pos.sible that damage to the lungs from breathing oxygen (Bean, 1945) might have
impaired the oxygenation of the arterial blood, either because of pulmonary
oedema or intrapulmonary shunting of blood. However, local hypoxia in the
tumour seems a more likely explanation than general arterial hypoxia.

Finally, a radioresistant strain of cells can be postulated, but this seems un-
likety (Conger, 1956 ; Nice, 1957).

dose experiment to produce matching results with differing

ed m'Tu conditions of oxygenation has been approximately achiev-

comparison is made between all tumours in each groun IFia 121
ofiowth following a common growth pattern (Fig. 1,3) two pairs

^re nearly the same. The effect of 4000 rads given to the annyir.

the rat bre£g

574

R. H. THOMLINSON

Since the whole growth pattern of the tumours lias been disturbed by any of
these forms of radiation, it may be doubted that the matchings of one groirth
curve yuth anotlier at any particular point is valid. Very good matches could
be obtained between all tlie curves within the first five post-irradiation days' It
therefore seems worth while to consider and later to investigate the factors wliicli
govern the shape of any of these curves.

Clearly the curves are composite, representing on the one hand the rate of
removal of dead tumour tissue, and on the other the multiplication of surviving
tumour cells. The curve of the rate of removal of dead tissue will also be com-
posite, because dead cells lying amongst capillaries with an active circulation
are eliminated very much more rapidly than a necrotic mass which has to be
reorganized. Both these processes are likely to be affected by the effects of
radiation on the capillaries.

A number of possibilities affect the shape of the curve representing multi-
plication of the tumour cells surviving radiation.

The linear relationship between the radius of the tumour and time in the control
curve suggests that cell death is talcing place in the larger tumours at about the same
rate as cell production. In these tumours the Avhole central region is necrotic
and is surrounded bj'’ a viable rim. If this rim maintains a constant thickness,
which approximately it does, its volume increases five-fold as the radius of the
tumour doubles. Since the time taken to double the radius is about four and a
half days and the number of intact tumour cells is proportional to the volume of
the rim, the generation time of these cells is slightly less than one day.

The curve of tumour size after irradiation with 4000 rads in oxygen shows a
doubling of the radius in twelve days, beginning on the fourteenth post-irradiation
day. This might indicate a generation time of two and a half days — an unlikel}’'
delay of metabolic processes in cells surviving the first two weeks. However,
the growth rate may be reduced by nutritional deficiency due to the effects of
radiation on capillary blood-vessels. Another possibility is that many cells have
suffered less than lethal genetic damage and that in a series of cell divisions dam-
aged material is gradually eliminated in non-viable daughter cells. In this wa,y
the total number of viable cells and therefore the tumour mass might remain

almost constant for a long period.

In the animals which surmve long enough for the tumour to reach the large
size of 50 mm. diameter the grovdh curves gradually steepen and come almost or
quite equal to the slope of the control group. The times taken to reach this size
are sho\vn in Table III. The longest time was 46 days after the dose of 4000 rads
in oxygen. This may be compared ■with the control groui^ which rea,ched mis
size 14 days after the treatment day and 29 days after implantation. The
difference between 29 days and 46 days might be explicable in terms of the number
of cells surviving the implantation process in the first case and the number sur
viving irradiation in the second. If this is so, the shape of curves representing t le
cell multiplication processes could be identical and the times taken by each group
to reach the large size would be proportional to the number of cells surviving,
is interesting that on this basis there is also a close match between t e group
recei-ving 4000 rads in anoxic conditions and 2000 rads in air, and t le
receiving 4000 rads in air and 2000 rads in oxygen. It will be of interest to
investigate the various possible factors influencing the shape of the curves.

These results confirm those of earlier workers with ‘ sohd tumours (Hole ,

METHOD FOR COMPARING TREATMENTS OF J UMOURS

Lorenz and Matthews, 11152; .Soott, 1953; Dittrich and Stuhimann, 1934 ;
Griissner, 1957; du Sanlt, Bylcr and Dohhen, 1959). IVhilst no great mathonni-
tical precision should he attached to the ratio of 4 : 1 shonn in the efTect on the
tumour Aiith the rat breathing oxj^gen compared ivith the anoxie tumour the

results do support tlu-ee conclusions : , , • r

1. In the tumour RIBo there are cells winch arc jirotected by anoxia Irom

radiation damage whilst the animal is breathing air.

2. After irradiation “ in air ” these cells arc capable of multiplying to

regenerate the tumours, and

3. The radiation injurj' to these cells is enhanced bj- giving the rat oxygen
to breathe at 45 lb. pressure.

The ph 5 ^siological and pathological mechanisms bringing about these effects are
equally liltely to apply in human tumours as in rat tumours u-hcrc the same
patterns of growth and circulator}" disturbance are found. It is therefore likely
that the use of oxygen in the radiotherapy of human cancer will diminish the
number of cancer cells surviving a given dose of radiation and increase the pro-
portion of patients cured.

S0M5IARY

A technique has been developed for growing transplantable malignant tumours
in the subcutaneous tissue of the rat in such a way that they remain localised until
they have grovTi to a suitable size — 10 mm. diameter — for testing the effects of
different treatments. The course of the tumours was followed in situ by daily
measurement. Comparisons have been made of the effects of single doses of
2000 rads and 4000 rads of 250 kv. X-rays under three different conditions of
oxygenation of the tumour ; with the tumour made anoxic by clamping the
circulation, vith the tumour “ aerated ” vith its circulation intact and the
rat breathing air at atmospheric pressure and vith the tumour “ oxygenated ”
with the animal breathing oxygen at 4 atmosphere’s pressure. The effect of
2000 rads given in air approximately equals that of 4000 rads to the anoxic tumour
and the effect of 2000 rads in oxygen approximately equals that of 4000 rads in
air. These results indicate that wdien the rat breathes air there are cells in the
tumour protected from radiation injury by h}q?oxia ; that after radiation in air
such cells can regenerate the tumour and their radiosensitivity can be enhanced
by the breathing of oxygen at high pressures. The pathological basis of these
conclusions suggests that they apply equally to many forms of human cancer

I should like to thank Sir Robert Davis for the gift of the pressure chamber
used m these experiments. Dr. J. B. West of the Post Graduate Iiledical School for
measurements wuth the mass spectrometer, Jlr. D. Moore for measurements of
radiation dose, my technician Jlr. J. Whitaker, and other members of this Unit
almost all of whom have helped to make the w'ork possible. ’

REFERENCES

COXOEK, A. D.— (1956) Radiology, 66, 6^

576

B. H. THOMLINSON

Dbschnee, E. E. and Gray, L. H. — (1959) Sadiation Ees., 11, 115.

Dittrich, W. and Stuhlmann, H. — (1954) Natunvissenschaften, 41, 122.

Gray, L. H. — (1958) ‘ Lectures on the Scientific Basis of Medicine ’. Voi. WI, 1957-58
p. 314.

Idem, Conger, A. D., Ebert, M., Hornsey, S. and Scott, 0. C. A. — (1953) Brit. J.
Badiol., 26, 638.

Grussner, G. — (1957) Strahlentherapie, 104, 514.

Hemhtt, H. B.— (1958) Brit. J. Cancer, 12, 378.— (1959) Ibid., 13, 675.

Holcroet, j. W., Lorenz, E. and Matthews, M. — (1952) J. nat. Cancer Inst., 12, 751.
Nice, C. M. — (1957) Amer. J. Boentgenol., 78, 831.

Prehn, R. T. and JIain, J. M. — (1957) J. nat. Cancer Inst., 18, 769.

Scott, 0. C. A. — (1953) In Gray, Conger, Ebert, Hornsey and Scott, q.v. — (1958)
‘ Advances in Biological & Medical Physics ’, Vol. 6. New York (Academic
Press). — (1960) Badiation Bes. In press.

DU Sault, L. a., Eyler, W. R. and Dobben, G. D. — (1959) Amer. J. Boentgenol.
82, 688.

Thomlinson, R. H. and Gray, L. H. — (1955) Brit. J. Cancer, 9, 539.

577

A STUDY OF DOSE AND EFFECT IN INITIATION OF SKIN
TIBIOURS BY A CAECINOGENIC HYTDEOCAEBON

J. K. BALL AND J, A. McCARTER

From the Department of Biochemistry, Dalhousic University,

Halifax, Nova Scotia, Canada

Received for publication June 29, 1900

In the study described in this paper, we have attempted to learn Iiow the
incidence of tumours elicited in mouse skin by initiating and promoting stimuli
(reviewed by Salaman, 1958) is related, quantitatively, to the amount of carcino-
genic hydrocarbon absorbed by the skin, rather than to the amount applied.

Methods have been devised (McCarter, 1956) to confine the applied hydrocarbon
to a measured area of the skin of an immobilized mouse and to limit the amount
absorbed by limiting the time that excess hydrocarbon remains on the skin.
Some animals are killed so that the amount of the hydrocarbon absorbed by the
skin can be determined. Others are allowed to live and are treated topicallj’- with
croton oil to produce tumours.

Using these techniques, it was possible to show (^IcCarter, Szerb and Tliompson,
1956) that the number of tumours produced in the skin of a mouse varied directly
with the area of skin covered by the hydrocarbon. Math the time allowed for
absorption and with the logarithm of the concentration of the solution applied
(9,10-dimethjd-l, 2-benzanthracene in liquid paraffin). Subsequently (McCarter,
1958), an attempt was made to relate the tumour incidence to the amount of
hydrocarbon (3,4-benzop3wene) that penetrated unit area of skin in a given time
following the application of a solution of the substance in acetone. The present
paper describes a similar, but more extensive investigation using 9,10-dimethyl-
1, 2-benzanthracene, hereafter called DMBA.

METHODS

Application of hydrocarbon to the skin

The animals used were female mice of the CFW strain (Carworth Farms Inc.,
New City, New York) 8 to 10 weeks of age. They were housed in groups of 10 in
acrjdic plastic boxes with stainless steel tops. The bedding was sawdust AVater
and Purina Fox Chow Cubes were freely available.

Each animal was immobilized by the intraperitoneal injection of a solution of
Meprobamate (Jbitown, kindly supplied by Dr. F. M. Berger, Wallace Labora-
tones, Nev Brunswick, N.J.) usmg 0-40 g. per kg. body weight. This was

oT ner^Ldv T 'V A^second dose of

Wre to be &I2il3feYK"

578

J. K. BALL AND J. A. McCARTEB

production, lack of hair growth during the next few days. This teclniique might
have allowed a few animals early in the groivth stage to have been included among
those used for the chemical analyses, but Ave vished to avoid any complications
that might arise by prior treatment of the skin, by plucking, for example.

A solution of DMBA in redistilled acetone Avas applied to a circular area of
2-3 ± 0-3 sq.cm. (Standard Deviation) on the mid-portion of the back using the
applicator described by McCarter (1956). The mouse was then placed until
required in a dark incubator maintained at 25° C. Then, the excess of hydrocarbon
remaining on the skin Avas carefully removed by Avashing Aidth diethyl ether
(McCarter, 1956). The animal Avas returned to its cage to aivait further treatment
AAuth croton oil, or Avas killed so that chemical analysis of the dosed area of skin
could be made.

Production of tumours

Beginning three Aveeks after treatment of the animal Avith DMBA each mouse
Avas painted tAAuce Aveekly Aidth 2-5 per cent croton oil (Bush and Co., Canada) in
liquid paraffin (paraffin oil, Fisher Scientific Co., Montreal, Canada ; Viscosity
125/135, N.F.) using an artist’s N^o. 6 camel hair brush. The solution Avas spread
over an area greater than that treated Aidth DMBA. The appearance of the back
of the mouse Avas recorded in a sketch once each Aveek (usually checked by another
obserAmr) so that the time of appearance and subsequent history of each tumour
could be noted. Any tumour larger than 2 mm. in diameter (approximately) and
observed on 3 consecutive AA'eeks was recorded.

Chemical analysis

The animal Aims killed by a bloAv on the head. The position of the circle
treated Avith DMBA Aims made visible by brief illumination Avith an ultraviolet
light having the major part of its emission at 366 my. The treated area of skin
AA'as excised together Avith a narroAV untreated margin.

A suitable number of such treated circles (5 for contact-times of 1 hour or
longer ; 10 for times less than 1 hour) Ai'^ere pooled and extracted in a Soxlileb
apparatus AAoth 95 per cent ethanol (80 ml.) for 24 hours. Under these conditions,
extraction of the hydrocarbon from the skin Avas complete. The ethanol extract
Avas heated under a reflux condenser Avith 2 ml. of 50 per cent potassium Itydroxide
for 6 hours to saponify lipids. After cooling, the mixture AV'as diluted Aidth water
and extracted repeatedly A\dth petroleum ether (b.p. 30 to 60° C.) in a specially-
constructed glass U-tube (so that contamination Avith grease et cetera could be
aAmided). The petroleum ether extracts AA’^ere combined and evaporated to dryness
under reduced pressure.

It AA'as necessary to separate the hydrocarbon from substances in the extract
that interfered AA'itli the measurement of the ultraviolet absorption spectrum.
The residue derived from the evaporation of the petroleum ether Avas dissolved in a
mixture of petroleum ether and benzene in the proportions 80 : 20 (v/v) and the
solution Avas transferred to a chromatographic column of Florisil, 9-7 g., 60 to 100
mesh in a tube 2-2 cm. in diameter. (Florisil, Fioridin Co., Tallahassee, Florida .
The solvent mixture was passed through the column and collected in m .
portions. Those that contained Di\IBA Avere located using ultraviolet absorpton
spectroscopy, pooled and evaporated to dryness under reduced pressure. e

IKITIATION OT SKIN TUMOUKS

residue Avas dissolved in 95 per cent ethanol and transferred quantitatively to a
suitable volumetric flask. The ultraviolet absorption spectrum of the sohi^tion
was measured relative to that of a similarly prepared control solution derived from
a quantity of skin equal to that used in the analysis and obtained from mice
dosed on the skin AAutli acetone and washed Avith diethyl ether. Silica cells having
a light path of 1 cm. Avere used in the Beckman DK-2 Ratio Recording Spcctro-
photometer. The absorption spectrum from 2C0 to 340 m//. provided qualitative
identification of DJIBA and the concentration Avas calculated from the molar
extinction coefficient at 297 m/i. (En,oi,ir 297 m/i. = 4-90).

Using the analjiiical procedure described aboA’c, amounts of DMBA A’arying
from 0-6 to 6-4 /ig. added to mouse skin AA-ere recoA-ered as noted in Table I.

Table I. — Analytical Recovery of Amount of DMBA Added to Mouse Skin

Added

Recovered

(/<g-)

(/'?•)

0-6

0-G

2-2

3-2

3-4

0-4

fi-3

6-4

fi-4

6-4

C-0

KESULTS

In experiments to determine the amounts of DilBA in the skin after AA-ashing
the excess from the surface, different results AA-ere obtained by different experi-
menters. The data of Table 11 illustrate these results. It is apparent that,

T-able n. — Amounts of DMBA in Skin of Mouse at Various Times After Appli-
cationof Solution 0-2o per cent in Acetone. Means of 4: Analyses, 10 Circles per
Analysis for Times Less Than 1 Hour ; 5 for Times Greater Than 1 Hour.
Standard Errors. Area, 2-3 sq.cm.

Time

Investigation B

Investigation

(hours)

(ns)

(ns-)

0-25

l'09i005

0-.3.5-L0-03

0*5

105±013

0-3.=;-h-0-08

. I-40.^-0-04

l-47±0-13

I-89-0-12

2-73.^0-2.5

2 09^006

3-73.^0-16

•

4-18i0-0G

tl.= .kin .ould Mnence it. «PPlW to

to iVpar™ t 'iiel t° S'S rf “"“"‘-tions O-OO.?

immobilized mouse (5 in each erounl ns" ^ ot the hack of an

Three hour. later, the excess of hydroearbo^ on rtove^anlX

J. K. BALL AND J. A. McCABTBR

treated areas were excised, pooled and analyzed for their content of Dj\IBA The
results of this experiment are recorded in Table III. It is apparent that the

Table lll.~Influence of Variation in Amount of DMBA Applied to Skin on
Amount that Penetrates in 3 Hours. Means of 4 Analyses, 5 Circles per
Analysis. 0-15 ml. Applied to 2-3 sq.cm. Standard Errors.

Concentration
of solution

applied

DMBA in skin

(%)

ins-)

0-005

7-5

0-50±0-07

0-01

0-81±0-18

0-05

3-48±0-09

0-10

150

2-44±0-05

0-20

300

l-87±0-03

0-30

400

l-38±0-20

0-70

1050

l-55dt0-10

1-0

1500

l-88±0-21

absorption of DMBA increased as the amount applied was increased from 7-5 to
75 yg., then decreased and became relatively constant when 300 to 1500 us. was
applied.

MTien an amount of 75 yg. DMBA was applied to the skin in 0-15 ml. of 0-05
per cent solution in acetone and the excess hydrocarbon was washed off at various
times thereafter, the results shoum in Table IV were obtained.

Table IV. — The Penetration of DMBA into the Skin After the Application of
75 yg. (0T5 ml. of 0-05 per cent Solution in Acetone) to 2-3 sq.cm. Means of
3 Analyses, 5 Circles per Analysis. Standard Errors.

Time

(liours)

0-25

0-50

DMBA in skin

(m-)

0-15±0-03

0-41±0-05

0- 89±0-07

1- 91±0-13

3- 48±0-09

4- 74±0-15

Tumour incidence

The data for the distribution of tumours among all the mice exposed to DMBA
are recorded in Table V. Also included in this table are the results obtained
using a control group of mice dosed with acetone, immobilized for 7 hours and
washed with diethyl ether. These mice, like tliose that had been treated vlth
DMBA, were painted twice weekly with croton oil. Data for the time to appear-
ance of the first tumour in a mouse (timed from the daj^ that treatment witli
croton oil was begun) are recorded in Table VI. Times to appearance of all the
tumours are reported in Table VII. A summary of all the data is given in Table
VIII. Fig. 1 and 2 are discussed in the next section.

DISCUSSION

Before undertaking this Avork, we thought that variation in the amount of
solid hydrocarbon applied to the skin, such as could be achieA’'ed by appymg

Table Y .—Distrihulion of Tumours Among Mice

INITIATION OF SKIN TUMOUKS

581

« Cl C5 O o
t- Cl 1** o -r

o i-n X X X
^ S3 l- t- X

X t Cl 1- — tc
Cl O C5 ^ -4* CJ

Cl Cl Cl cc

X Cl Cl X «

Cl Cl Cl Cl Cl Cl

t- C ^ CC Cl
ec I'* I'- f o

X m -I* X X

X C -?• Cl

Cl tn c: -f X o
»n X Cl f X

^ Cl Cl «

Cl X Cl Cl Cl

— -H Cl Cl X X

in t'- Ci

1- c: cn

X — Cl 1- C5

X 1^ Cl m Cl

in o X X X c:
X o I'* — -r

m t-* m « in
m m in »n »n

S3 sn o t- X
m m U5 m m

X -t X Ci

in «n I'* i'“ X X

• • • • •

• ■ ■ -

; ; :

• • 'Cl —

;

; :

• • • Cl Cl

■ -ri • ■ ■

• — — * Cl

rt ;

• . Cl • . .

*— • • Cl ^

. Cl » — .

. . Cl ^ ^

• — Cl Cl Cl

Cl Cl — ^ Cl cl

. ^ Cl ^ ^

Cl Cl — W .

• ■ — — 'C Cl

Cl CC Cl Cl

-4* Cl •4' • •

Cl Cl Cl X Cl X

• ^ sc -4- O

X « •4' Cl

— Cl X -4- X X

<— -4* m .

• C5 X in X

X m t-* Cl X in

l> -4* -t -!•

'»4* cc X X »n

*4* X X X X in

c; sD CO 00 in

-4- O LC -4* C5

»n C5 — <r. Cl

-t X — C5 ^

«n X X X C5

o in o X -* X

Cl sn -4* C5 cc
Cl Cl ^ ^

l> O X -+ -"t
Cl — Cl Cl

sn sn m t- in tn

Cl — Cl Cl

m C5 X -4*
-4* X -4* t-

X m o X c:
o o X o

•n m t- X X X
X X •c i-* l- —

O O O X •«4‘

»-H ^ , Cl

O c — Cl X -*■

m o

Cl o

m o

Cl m

m o

Cl m

o o 1-4 cc in

o o ^ X in

o o X in i-

LC ^

' >n
o t-

m o

Cl X

6cq

o ns
o o
m ^ ;n

Cl ^

Table VI. — Time to Appearance of First

J. K. BALL AND J. A. McCARTER

i t- O CO 1C

ic ic »c

o CO O l> CO CO
»0 to U5 to lO CO

CO rj* CO ^ rt* C5

to to l> t- CO CO

CO *-1 o Ci
eo CO 'Tf CO CO

OCOC^lcOH'rt-
<M 'i* CO CO CO M

t- CO O t'- C5 CO
03 CO f 03 CO

: : :^ ; :

CO ♦ • . .

. . . c>5

• 03 . ^ ^

* 03 -H . . .

. ♦ (M .

: ;

. -CO • • r-*

M .

^ . P-< r-l

^ CO * r- *

(M ^ -H

— 1 03 03 • 1-H

<M <M

^ W 03 — • •

^ '0 03 03 •

. — * ^ CO

CO CO 03 CO 01 03

03 CO 03 --H ^ ^

CO CO t>* —

— 03 03 ^

-- 03 CO -ri* 03 03

r' Lo CO CO to

CO r» to CO CO

CO 03 03 —

Ct 00 c: c: lO

00 to — * r» c:> CO

03 03 CO CO CO O

•*+ to to o

CO O CO

ta CO CO O CO

W to Tf (M

«M . Tf- 1-0 to

^ ta r- to CO

; I"'

I-H - CO 1— < -<

>-f CO ' CO ‘CO

'*11!

• • • » • ^

: ;

lO — Ct 00 »o
»-i tJ* CO

o: to C5 C5

O O CO o •

la to CO CO CO

CO CO -«*• r- 1> ^

O O O CO

<-H ^ rM 03

O O 03 CO -0^

to o

OJ to o o o

to o ^

03 ta o o o o

to o

03 to O O O O

O O CO »o

o o ^ CO to r*

0-25

Table VII . — Time to Appearance of All Tumours Dated f tom 1 ime

INITI.NTIOX or SKIN TVMOVUS

584

J. K. BALL AND J. A. McOARTER

Table Ylll.—Bata of Tables II and IV to VII Rearranged to shoiv Variation in
Emrrale7lwim Variation in DMBA in Shin. Standard

D5IBA in skin
(t'S-)

0-00

0-I5±0-03

0-35±0-03

0-35±0-08

0-4l±0-05

0- 89±0-07

1- 05±0-13

l-09±0-05
1-40±0-04
l-47±0-13

1- 8a±0-12

2- 09A:0-06

2- 73±0-25

3- 48±0-09

3- 73±0-10

4- 18±O-0Q

4-74±0-I5

Latent period

Tumours

Number

First tumour

—A

All tumours

per mouse

0-09

of mice
90

(weeks)

1-3G

10-85:t0-57

n-92±0-39

1-52

1-8G

8-37±0-57

9-29±0-35

9-84iO-G3

10-84±0-34

1'70

10-35±0-45

ll-80±0-36

2" 71

10-09±0-42

Il-56±0-40

3-06

9-45±0-3I

10-7S±0-18

2-38

9-86±0'43

I0-92±0-2C

2-44

9-91±0-44

Il-23±0-28

2-49

10-32±0-42

n-07±0-2I

2-76

10-03±0-46

ll-0S±0-23

2-23

0-80±0-42

10-67±0-23

2-23

8-S7±0-29

ll-05±0-34

2-43

10-85±0-43

12-07±0-24

3-47

9-28±0-44

10-61±0-26

3-82

9-4S±0-50

lO-GGiO-lT

3-02

8-77±0-36

n-05±0-34

varying volumes or concentrations of a solution in a solvent that readily evapo-
rates, siiould be v'itliout effect on tiie amount of the h 3 ’’drocarbon absorbed bj’^
unit area in a given time, provided tiiat enough was applied to maintain a saturated
solution in tiie sebum. The latter condition should ensure the absorption of the
hj'^drocarbon at the maximal rate. On this basis, the increased absorption of
DMBA Avhich resulted Avlien the amount applied Avas increased from 7-5 to 75 /tg.
(Table III) may be explained b}^ assuming that amounts less than 75 /ig. Avere not
enough to prOAude a saturated solution in the sebum. A different explanation has
to be sought for the fact that the absorption decreased as the amount applied AA'as
increased from 75 to 300 /ig. and thereafter became relatiA^^ely constant. It seems
probable that, as the hulk of the solid hydrocarbon Avas increased, it soaked up
some of the sebum, particularly that readily aAmilable on the surface of the skin,
as indeed anj'" poAA^dery substance should do. The importance of sebum for the
absorption of DMBA aa'us demonstrated in an experiment in AAdiich the skin was
AA^ashed Avith diethyl ether before the hj’^drocarbon aa^s applied. Under these
conditions, only 0-66 /eg. of DMBA AA-as absorbed in 2 hours as compared Avith
1-91 /eg. in the same time by unwashed skin.

The differences obtained by the tAAm experimenters aa'Iio measured the pene-
tration of DMBA at Amrious times (Table II) must be attributed to the fact that
the quantities applied to the skin AA^ere different in the Iaa^o experiments. One
investigator (B) applied approximately 300 /eg. or more ; the other (H) applied
only 100 to 150 /eg., both in the belief (supported by our earlier experience AA’ith
3,4-benzop3Tene— McCarter, 1956) that Amriation in the amount applied Avas AVith-
out influence on the amount absorbed. The values recorded in Table II for ■
hours of penetration are consistent AAu’th predictions made on the basis of le
amounts applied and the data of Table III. HoAA'eAmr, the data of Tables II anc
IV shoAV that a greater initial penetration of DMBA took place AA'hen the amouu ■
applied AA'as increased. Thus, in 0-25 hours, 0-15 /eg. of 75, 0-35 of 100-15 , anc

INITIATION or SKIN 'll’MolT’.S

line fitted by the inetbml of l-nM M.tmr, - bn. tbn .....m,.,,,, v
' I’l- log (10 X //g. DMBA). Stnndnnl error of the 1.1(1]... (i-SI. '

O.r

I n

586

J. K. BALL AND J. A. i\IcCAETER

^■'^"tors governing the absorption
tobe^^"^ ™ouse skin must be more complex than we have supposed tliem

The data of Table IV were obtained liaving regard for aU the known variables
The data are fitted by the straight line F = 0-004 + 0-96 (hours of contacti
where J = /<g. of DMBA per dosed circle (2-3 sq.cm.). The standard error of
tlie slope was 0-01. The fact that the rate of absorption of the hydrocarbon
v'as constant during the 5 hours’ period under consideration suggests tliat there
was no ma.rked alteration in permeability of the skin during that time.

The mistake made in assuming that variation in the amount applied ivoiild
have no influence on the amount absorbed, would have made it impossible to
compare the data on tumour incidence obtained bj'^ the two investigators, and
certainly it would not have been possible to combine the results as has been done
in Table VIII, but for the fact that the amount of DMBA in the skin was measured,
thus providing a common basis for comparison. It was important to know that
each set of experiments had been performed rvith a high degree of reproducibility
(standard errors. Table III) so that reliance could be placed in the corresponding
anal 3 ’tical and tumour data. It was possible, therefore, to use aU of the data.

Tlie information provided by these experiments was examined for the existence
of relationships between the amount of hydrocarbon in the skin (disregarding the
time for absorption as a factor) and the tumour jaeld. For each group that had
received a given dose two measures were available ; one was the average number
of tumours borne bj'' the animals that survived the experiment : the other was
the proportion of tumour-bearing mice relative to the number of survivors. It
must be stressed that the number of tumours recorded per mouse was the number
of new tumours produced during the whole period of 20 weeks and was not the
incidence at any particular time. The index we have used is independent of the
balance struck between the rate of appearance of new tumours and the rate of
disappearance of old (discussed by Salaman, 1958). We have not tried to do any
calculations relating the rate of regression of tumoims to the dose of DMBA (the
amount in the skin) because we are uncertain about how to score a tumour as one
regressing or ha^fing regressed.

The amount of hydrocarbon found in the skin bj'’ analysis is less than that
which has penetrated because some is metabolized or transported from the site.
The rate of loss of DMBA left after washing off the excess and measured over the
first twelve hour period foUouing absorption, has a half-life time of the order of
10 hours (Huh and McCarter, 1960). This value maj’’ be used to estimate, for
example, that during the 7 hour period at the end of which 4-74 /tg. DIMBA was
present in the skin, 6-2 must have been absorbed, of which 1-4 had been metabo-
lized or transported. This value is very much smaller than that estimated by
Booth and Boutwell (1959) who attributed the difference between the amounts ot
DMBA applied to the skin and recovered later at the site of application m restrained
animals, to absorption through the skin. The rates of accumulation an is
appearance measured by us are not consistent vdth the assumption made by lese
authors. The correction for loss during absorption was not applied y us in

assessing the dose-response relationships. . ,

The data of Table VI were analyzed using the method of probits as descnueu
by Finney (1950). There was no evidence that the results could be descriDcu
by a straight line having the equation T = 5-17-f0-18 log ( 1 0 X /ig. Di IB ms

IXITIATIOX OF SKIN* TUMOUKS

.587

produced per mou J by a factor of 1.5, Avhcrcas a further .ncrease o .)• , .1 ± d- 1 o
[factor 0^30, or factor of 40 if the value is eorrected for mclahohc loss) onl\
doubled the tumour peld. An analysis of variance of the data for the elTect ()f
variation in log dose on the number of tumours produced iH>r tnou^se
out according to .Snedecor (1946). There was a highly sigiuh^nt (/ < ^ )

regression of tumour jdeld on the logarithm of the amount of D.AIBA in the .skin.
The experimental results are described by the equation 1 umours ]icr mouse
M3 -L 1-12 log (10 X /<g. DAIBA in skin). The standard error of the slojie was ()■:.].
It would be incorrect to assume either that this relationshi]) is the only one eajiable
of describing the data or that it can be used to extra]iolatc much beyond the
limits of the data. One cannot decide whether or not the tumour yield coiit inues
to increase or if it approaches some limiting value as the amount of DAIHA is
extrapolated to higher values, though it seems apparent that the resjion.ce is
virtually saturated for amounts of DAIBA in the skin greater than 1 or 2 //g. jkt
dosed circle (about 0-5 to 1 /ig. per sq.cm.). We have no cx])lanation to ofl'er for
this apparent approach to saturation unless, perhaps, this observation represents
the early onset of the phenomenon recorded by Shubik and Ritchie (19.53) as a
failure to obtain summation of effects of multiple doses of DM BA. fsiinilarly, it
is not possible to decide if there is, or is not, a threshold dose for the initiation of
tumours, though it seems clear that if such a dose exists it must be ver^’ small
indeed.

If the data of the groups (rows) of Table V are plotted with numbers of animals
as ordinates and numbers of tumours borne by them (0, 1, 2, 3, etc.) as abscis.sae,
it is learned that the distributions are J-shaped. It might have been expected
that the data should conform to a Poisson distribution, but there arc veiy signifi-
cant deviations of the observed values from the expected, particularly for animals
having no tumours and for those having many tumours. In both instances, the
numbers obser^-ed far exceed the numbers predicted. We attempted to anah*7e
our data by the methods described by Polissar and .Shimkin (19.54) but without
conclusive result. The range of responses observed by us (1-3G tumours per mouse
with the lowest dose and 3-02 nith the highest) was too narrow to permit a trend
to be seen when the standard deviations were plotted against the means. For
eve^- group the obsen^ed standard deviation was greater than the calculated
(.S.D _ 7«. for a Poisson) but it was not possible to obtain evidence for or afrainst

that the deviations from a Poisson distribution were due
IL t’umoum^'SoPw''"^ interaction betwtn

might alio,

dieted on a random basS ^ frequenej^ than would be pre-

experiments, a^Fngle dbsl^may ^aV to Tpilat-o^^'^ of being washed off as in our

(Orr, 1938) and may result in the prod^c£ nf r skin

(Englebreth-Holm and Iversen 1951) to ^'ithout further treatment

, 1951). to our expenments, 31 CPW female

mice

58S

J. K. BALL AKD J. A. McCARTER

that had, initially, 4-2 /ig. of DilBA in the skin failed to develop any tumours in

o!? P®«od during which they were not treated^with croton

oil. ^ e liave not observed m our animals any loss of hair at the treated site in
the three weeks interval between the time of the limited exposure to DMBA and
the start of treatment with croton oil. On the other hand, when u^e left the hydro-
carbon on the skin, epilation and necrosis were always observed. More detailed
mv^ti^tion IS needed, but these observations suggest that initiation of tumours
by OMJ3A may be achieved noth a minute amount of hydrocarbon that does not
produce apparent injury to the skin ; injury results from the penetration of a
larger quantity of the hydrocarbon, but the number of tumours initiated is not
thereby necessarily increased. This is not to imply that injurj'- is not important
in the overall process of tumour production, but that for DMBA applied to skin,
as for urethane (Roe and Salaman, 1954) and triethylene melamine (Roe and
Salaman, 1955) and for DMBA by mouth (Berenblum and Haran-Ghera, 1957)
initiation of skin tumours may be dissociated from injury’-.

Altogether, 2510 tumours of the skin were recorded in this study. Judged by
their appearance, growth-rate and invasiveness (or lack of it), only 2 were malig-
nant and the remainder ivere papillomas. Of 655 tumours in 280 mice treated
with 75 //g. DMBA applied to 2-3 sq.cm, only IS (2-7 per cent) arose outside this
area. These were situated on the flanks or near the base of the tail but were always
close to the treated circle. Contamination of the skin around this area with a
minute amount of DMBA during the washing procedure could account for the
distribution of these tumours. The chance of contaminating tlie skin was greater
when the applied quantity of DMBA was greater. Of 1855 tumours produced in
638 mice dosed with 100 to 300 /ig. 235, or 12 per cent arose outside, but near, the
treated area. Tumours that ivere obviously outside the treated areas were
neglected in compiling Tables V to VIII.

A possible source of error in these experiments could arise from tumours
growing close together so that they might be indistinguishable from one another.
Tumours were first recognised when they had an area of approximately 1 sq.mm,
or about 0-5 per cent of the area in which they could appear. Even in the groups
having the highest tumour incidence, tumour-bearing animals had an average of
4-3 each, with a maximum of 15 so that the area occupied when the tumours were
first recognisable was a negligible proportion of the total available. Grovdh of
the early-appearing tumours might have interfered ivith the recording of those
appearing late but this could only have been a problem in the animals bearing a
large number of tumours, for example, 10 or more. Such animals accounted for
only 14 per cent of all tumours produced. Furthermore, most of the tumours
(80 per cent) appeared within a period of time (7th to 13th weeks) when all were
small and least likely to overgrow one another. It is not Ifliely, therefore, that
inability to distinguish tumours wms a major source of error in these experiments.

The latent period, dated from the time treatment with croton oil was begun to
the time of appearance of the first tumour in a mouse, or averaged for all the
tumours appearing in a mouse, did not change in a s/steinatic way wdh varmtion
in the amount of DMBA in the skin. Inspection of Table VII and Fig. 2 rai ht
suggest that groups of tumours arose characterized by different latent Penods
indLting perhaps that the tumours were of more than

support to the suggestion made by Shubik, Baserga and R^^^^^ie (19o3) tha
tumours produced by initiating and promoting stimuli m mouse skin differ over

INITIATION OF SKIN TUMOURS

osn

a range of gro^^iill potential. There is, however, no reason to suppose tliat the
fluctifations we have observed are other than random variations due to cliancc
Perhaps real differences would have been observed had tlic aniina s liccn Ivept for
a longer time, but we decided to stop the experiment wlien, as shown in Jog.

the tumours stopped appearing. *•

The methods described in this paper should be eapable of furnishing cstimale.s
of the relative initiating potencies of different liydrocarbons. We have found ly
analysis that a unit area of CPW mouse skin absorbs in 1 hour 1-4 x 10- moles
of 3 4-benzop}’Tene and 5-5 X 10"® moles of DMBA, after application of acetone
solutions of the hydrocarbons. Tlie corresponding tumour incidences were O-R
tumours per mouse for 3,4-benzopyrene and 2-44 per mouse for DMBA. Mhis tlic
number induced by DilffiA greater than that induced by 3,4-bcnzo]nTene merely
because there was more DjNEBA in the skin? Tlie question cannot be answered
without knoudng more about the dose-response relationsliips for tlie two liydro-

carbons.

Earlier work ndth 3,4-benzopjTene (McCarter, 19.58) in mice of the strain 1,
suggested a pattern of dose and response similar to that recorded here. An
attempt to use 3,4-benzopyrene in the CFW mouse resulted in the production of
too few tumours to allow conclusions to be drarni with confidence, but the data
are approximately described by the equation. Tumours per mouse = 0-25 -f 0-04
(moles X 10'® BP/sq.cm.). The rate of penetration of benzopjwene into CFW
skin did not differ significantly from that in I skin. These data allow a rough
estimate to be made of the relative sensitivities of tlie two strains of mouse. For
example, 2 x 10"® moles of 3,4-benzop3Tene per sq.cm, produced PS tumours per
mouse in strain I compared with 0-3 in strain CFW. The same dose of DMBA
produced 1-9 tumours per CFW mouse. Apparent^, the strain I mouse is about
6 times as sensitive as the CFW, and DMBA is 6 or 7 times as potent an initiating
agent as 3,4-benzopyrene. However, these estimates are only approximate because
in the CFW mouse, the only parts of the dose-response curves for the two hydro-
carbons that overlap involve the highest doses of 3,4-bezop5uene and the lowest
of DMBA ; no point was obtained at which the tumour yields were identical and
it is not possible to decide if the ratio of responses is independent of dose. One
would wish to compare the curves over the regions where response is most markedly
dependent on dose rather than where virtual saturation of the biological effect
is achieved but this would involve one in the measurement of smaller doses and
responses \sdth larger errors than were encountered in the present work. Had the
present experiments been done on a smaller scale or vith less rigorous control of
the variables concerned, it is probable that we should not have been able to draw
even the limited conclusions that we have dravm about the nature of the dose-
response relationships.

(DMBA) was applied in acetone to 2-3
sq.cm, of the skin of immobilized mice. After a time, the skin was washed and
some of the treated animals were kiUed for analysis of the DMBA content of the

688

J. K. BALL AKD J. A. McCARTER

that had, initially, 4-2 /tg. of DMBA in the skin failed to develop any tnmom in
the subsequent 20 weeks’ period during which they were not treated with croton
® observed in our animals any loss of hair at the treated site in

the three weeks’ interval between the time of the limited exposure to DlIBA and
tlie start of treatment with croton oil. On the other hand, when we left the hydro-
carbon on the skin, epilation and necrosis were always observed. More detailed
investigation is needed, but these observations suggest that initiation of tumours
by DMBA may be achieved with a minute amount of hydrocarbon that does not
produce apparent injury to the skin ; injury results from the penetration of a
larger quantity of the hydrocarbon, but the number of tumours initiated is not
thereby necessarily increased. This is not to imply that injury is not important
in the overall process of tumour production, but that for DMBA applied to skin,
as for urethane (Roe and Salaman, 1954) and triethylene melamine (Roe and
Salaman, 1955) and for DMBA by mouth (Berenblum and Haran-Ghera, 1957)
initiation of sldn tumours may be dissociated from injury.

Altogether, 2510 tumours of the skin were recorded in this study. Judged by
their appearance, growth-rate and invasiveness (or lack of it), only 2 were malig-
nant and the remainder were papillomas. Of 655 tumours in 280 mice treated
with 75 pg. DMBA applied to 2-3 sq.cm, only 18 (2-7 per cent) arose outside this
area. These ivere situated on the flanks or near the base of the tail but were always
close to the treated circle. Contamination of the skin around this area •with a
minute amount of DMBA during the washing procedure could account for the
distribution of these tumours. The chance of contaminating the skin •ivas greater
when the applied quantity of DMBA was greater. Of 1855 tumours produced in
638 mice dosed with 100 to 300 gg. 235, or 12 per cent arose outside, but near, the
treated area. Tumours that were obviously outside the treated areas were
neglected in compiling Tables V to VIII.

A possible source of error in these experiments could arise from tumours
gro'wing close together so that they might be indistinguishable from one another.
Tumours were first recognised w'hen they had an area of approximately 1 sq.mm,
or about 0-5 per cent of the area in rvhich they could appear. Even in the groups
having the highest tumour incidence, tumour-bearing animals had an average of
4-3 each, with a maximum of 15 so that the area occupied when the tumours were
first recognisable was a negligible proportion of the total available. Growth of
the early-appearing tumours might have interfered with the recording of those
appearing late but this could only have been a problem in the animals bearing a
large number of tumours, for example, 10 or more. Such animals accounted for
only 14 per cent of all tumours produced. Furthermore, most of the tumours
(80 per cent) appeared ■\^dthin a period of time (7th to 13th weeks) when all 'n ere
small and least likely to overgrow one another. It is not likely, therefore, la
inability to distinguish tumours Avas a major source of error in these experiments.

The latent period, dated from the time treatment ivith croton oil was
the time of appearance of the first tumour in a mouse, or averaged for a e
tumours appearing in a mouse, did not change in a systematic way wd varia i
in the amount of DMBA in the skin. Inspection of Table VII and Fig. 2 mi b
suggest that groups of tumours arose characterized by different latent perioa ,
indicating perhaps that the tumours were of more than one sort, and e „
support to the suggestion made by Shnbik, Baserga and ^ ,.fr 2 4r

tumours produced by initiating and promoting stimuli in mouse skin ditter

IKITIATIOK 01'' SKIN TUMOUKS

oS!)

“hS"Sis „e. sKo„W be ca,.eblc of fun.iel.i,,, c.(.in,nU.s

of the relative initiating potencies of different I'e

analysis that a unit area of CFW mouse skin absorbs in 1 lioiir 1 •• X 1 0 n oil s
of 3 ibenzops^ene and 5-5 X 10 -« moles of DMBA, after appl.ent.on of acetone
solutions of the hydrocarbons. The corresponding tumour >ncidcnco.s were -.1
tumours per mouse for 3,4-benzop>Tene and 2-44 per mouse for DMBA. as ( ho
number induced by DMBA greater than that induced by 3,4-bcn'/.oiiyrene merely
because there was more DMBA in the skin? The question cannot, be answered
without knoudng more about the dose-response relationships for the two hydro-
carbons. . .

Earlier Avork AAuth 3,4-benzopjTene (McCarter, 19.58) in mice of the strain 1,
suggested a pattern of dose and response similar to that recorded licrc. An
attempt to use 3,4-benzopjTene in the CFIV mouse resulted in the product ion of
too few tumours to allow conclusions to be drawn with confidence, but the data
are approximately described by the equation. Tumours per mouse = d-2r) 4- (b(»4
(moles X 10'® BP /sq.cm.). The rate of penetration of benzopyrene into CFW
skin did not differ significantly from that in I skin. Tliese data allow a rough
estimate to be made of the relative sensitivities of the two strains of mouse. For
example, 2 X 10'® moles of 3,4-benzopjTene per sq.cm, produced I -8 tumour.s jicr
mouse in strain I compared with 0-3 in strain CFIV. Tlie same close of DJI BA
produced 1-9 tumours per CFW mouse. Apparently, the strain I mouse is about
6 times as sensitive as the CFW, and DMBA is 6 or 7 times as potent an initiating
agent as 3,4-benzopyrene. However, these estimates are only approximate because
in the CFW mouse, the only parts of the dose-response curves for the two h^'dro-
carbons that overlap involve the highest doses of 3,4-bezop3Tene and the lowest
of DMBA ; no point Avas obtained at AA'hich the tumour 3 delds Avere identical and
it is not possible to decide if the ratio of responses is independent of dose. One
Avould Avish to compare the curves over the regions Avhere response is most markedly
dependent on dose rather than where virtual saturation of the biological effect
is achicAmd but this would inAmlve one in the measurement of smaller doses and
responses Aidth larger errors than were encountered in the present Avork. Had the
present experiments been done on a smaller scale or Avith less rigorous control of
the variables concerned, it is probable that Ave should not have been able to draw
even the limited conclusions that Ave have draAAii about the nature of the dose-
response relationships.

OU4»li>liLrVX

produce tumours. " topically with croton oil to

applied and rtlmtii^aU^werforTb^i dependent on the amount

590

J. K. BALL AND J. A. McCABTER

3. The number of tumours borne per mouse was a linear function of the loga-
rithm of the amount of DIVIBA in the skin.

4. Animals bearing no tumours, and those bearing many, were more numerous
than predicted by a Poisson distribution.

5. The time from the start of treatment with croton oil to the appearance of
the first tumour, or averaged for all the tumours in a group, appeared to be
independent of the amount of hydrocarbon in the skin.

6. Difficulties in the way of obtaining relative potency ratios of different
hydrocarbons are discussed.

The authors wish to thank j\Ir. T. Y. Huh for technical assistance and Dr. M.
Blackett for helpful discussion. We are grateful to the National Cancer Institute
of Canada for a grant of funds.

REFERENCES

Bebenblum, I. AND Haban-Gheba, N. — (1957) Brit. J. Cancer, 11, 85.

Hem AND Shubik, P. — (1947) Ibid., 1, 383.

Booth, B. A. and Boutweld, R. K. — (1959) Cancer Bes., 19, 79.

Engelbbeth-Holm, j. and Ivebsen, S. — (1951) Acta path, microbiol. scand., 29, 77.
Finney, D. J. — (1950) in ‘ Biological Standardization ’. J. H. Bum, D. J. Finnej" and
L. G. Goodwin, 2nd ed. London (Oxford University Press).

Huh, T. Y. and McCabtek, J. A. — (1960) Br/t. J. Cancer, 14, 591.

McCabteb, j. a. — (1956) J. nat. Cancer Inst., 17, 399. — (1958) Acta Un. int. Gancr.,
15, 178.

Idem, SzEEB, J. 0. and Thojibson, G. E. — (1956) J. nat. Cancer Inst., 17, 405.

Oee, j. W.— (1938) J. Path. Bact., 47, 495.

PoLissAK, M. J. AND SuiMKfN, M. B. — (1954) J. nat. Cancer Inst., 15, 377.

Roe, F. j. C. and Salajman, M. H. — -(1954) Brit. J. Cancer, 8, 666. — (1955) Ih\i., 9,
177.

Salaman, M. H. — (1958) Brit. med. Bxdl., 14, 116.

Shubik, P., Basebga, R. and Ritchie, A. C.— (1953) Brit. J. Cancer, 7, 342.

Idem AND Ritchie, A. C. — (1953) Cancer Res., 13, 343.

Snedecok, G. W.— (1946) ‘ Statistical Methods Applied to Experiments m Agriculture
and Biology ’. 4th ed., Ames, Iowa (The CoUegiate Press, Inc.).

PHENANTHRENE AS AE AETI-INITIATING AGliiNJ

TAI-YOUKG huh and J. a. HcCARTEH

From (he Department of Biochemistry. Dathnusic Unircrsily,
Halifax, Kora Scotia, Canada

Received for publientioii Juno 2!), HXiO

Ix 1945, Lacassagne, Buu-Hoi and Rudali reported that several weakly
carcinogenic substances when painted on mouse skin alternately with -0-me n
cholanthrene reduced the ability of the latter substance to iiroducc (uinours
This anti-carcinogenic effect was attributed to competition between the weak ami
strong carcinogens for some substrate within the cell because of their analogous
structure.

In the foUouing year, Crabtree (1946) showed that the non-caremogeme
hj’drocarbons, anthracene, naphthalene and especially ])hcnanthrene ]>osses.«c{l
anti-carcinogenic activity when tested in multiple applications against 3,4-bcnzo-
pjTene and 20-methylcholanthrene on the skin of mice. Investigations rojiortcd
by Riegel ei al. (1951) using 20-methylcholanthrenc and by Hill e( al. (1951)
using 9, 10-dimethjd-l, 2-benzanthracene showed that several hydrocarbons
depressed carcinogenic potency but others enhanced it.

In all of the above studies multiple applications of the hydrocarbons were used,
thus making it difficult to decide at what stage in the process of tumour jiroduction
the effect of the anti-carcinogenic agent was exerted. We tried to simplify flic
e.xperiment by testing the effect of a single application of phenanthrene on the
peld of tumours induced bj’- a single application of 9,1 0-dimethyl-l, 2-benzan-
thracene (DMBA) and multiple applications of croton oil. Some of the methods
used have been described previously (McCarter, 1956 ; McCarter, Szerb and
Thompson, 1956 ; Ball and McCarter, 1960).

EXFERIJIENTAL

The hydrocarbons 9, 10-dimethyI-l, 2-benzanthracene and phenanthrene (East-
man Kodak Co., Rochester, New York) were purified by chromatograT)hv on
Rlonsil (60-100 mesh, Rloridin Co., Tallahasee, Florida) and were checked for
punty by measurement of the ultraviolet absorption spectra. Solutions were
prepared by dissolving the substance in acetone to make mixtures having the
compositions shoum m Table I. ^

® ® distilled before use. Cyclohexane was further purified bv
jS. 1 cSlSi! ^ Sdentilic Co!,

The animals used, "were female minp nf tlif* n’K'nr o ^

applications of cS ^ ° ""''“P''

McCarter, 1900). As in those eape^SS so S tL

for the pro^nction of tn.onrs t oLrs .er; ^^tSS

592

TAI-YOUNG HUH AND J. A. McCARTER

Pem^tence of DMBA and Phenanthrene in Shin of
CPH jmce. 0-15 ml (^Acetone Solution Ajiplied to Oirck 1-64 to 1-66 sq cm

Hydrocarbon in skin at follomng times after application

Solution

(/ig. per sq. crn.)

applied

(%)

DMBA 0-2
Rhennnthrene —

0-S hour
. 0-16i0-08

1 hour
0'6o±0-13

3 hours
I-2I±015

6 hours
0-87±0-05

9 hours
0-70±0-07

15 lioms
0'35i0-04

~2?

hours

0-0

DMBA 0-2
Phenanthrene O'Oo

. 0-49±0-05

. l-00±0-25

l-12i0-07

l-66±0-18

2-27±0-41

3'05dr0*55

1-38±0-05

1-OG±0-14

M6±0-l

0-52±0-04

0-34-O-Oj

0-0

DMBA 0-2
Phenanthrene 0- 1

. 0-82±0-05

. 0-62±0-04

1-05±0-06

0-87±0-37

l-68±0-33

3-39±0-46

l-57±0-14

l-20±0-2S

l-09±0-18

0-67±0-2]

0-914-0-05

0-0

DMBA 0-2
Phenanthrene 0-2

. 0-75±0-00

. l-24±0-07

0- 94±0-09

1- 49±0-18

0-87±0-I7

3-22±0-19

0- 78±0-33

1- 66±0'26

0-47 + 0-04
0-72±0-l

0-0

DMBA —
Phenanthrene 0-2

! l-29i0-22

l-89±0-38

6-42±0-17

2*44i0-26

0-08±0-05

0-0 "

0-0

Excess hydrocarbon washed off skin after 3 hours.

circle of skin could be excised and analj^ed for its content of li 3 ^drocarbons. In
order to measure the persistence of the hydrocarbons once they had penetrated
the skin, still other animals were killed at intervals up to 24 hours after washing
off the excess of hydrocarbon and the dosed circles of skin were then taken for
anatysis. Pertinent details will be found in Table I.

Analytical method

Ten dosed circles of skin obtained from 5 mice were weighed together and were
then placed in a Soxhlet apparatus where thej’^ were extracted for 48 hours with
approximately 80 ml. of 95 per cent ethanol. To the extract, 2 ml. of 50 per cent
potassium hj^droxide was added and the mixture was heated under a reflux
condenser for 4 hours to saponif}'' lipids. The mixture was then cooled, diluted
Avith 40 ml. of water and was repeatedly extracted udth cj’^clohexane. For this
purpose, it was found convenient to divide the mixture into two parts and to
extract each separately in a glass U-tube using 10 ml. rmlumes of cj^clohexane four
times. The pooled extracts rvere then transferred to a column of Florisil, 60-100
mesh, 35 g. 2-2 cm. in diameter and 14 to 15 cm. long, for chromatographic

separation of the two hydrocarbons. j i an i

Phenanthrene Aims contained in the fractions collected betAA^een 80 and 8 m .
and Avas completely eluted from the column by cj^lohexane. Preliminary es s
had shoAAm that DlilBA did not appear until 220 ml. had run through the column
and it could not be completely removed by cyclohexane. Therefore, as soon as
the phenantlmene had been collected, the addition of cyclohexane to tie co umn
Avas stopped and a mixture AA'as substituted consisting of benzene and pe ro euin
ether (b.p. 40-60° G.) in the proportions 20 : 80 (v ; v). The remoAm o i
from the column began at once and Avas complete AA^hen approximate y
eluate had been collected.

PHEKACTHMKE as ax AXTMKlTIATIXn AfnvNT

Beca.se of rt,e vclatUify of ^ "Sit;' f

hence no reduction in volume conk ^ic llas]<.

phenanthrenevere, and cvaiioratcd to dryncKs under

« i. on cent, efl.a.o, an.,

'■"'ZTsotatlptotrif t,.^ solutions were .ncnsure.l relative I o those of

eorTtrolpreparaHonsder^edta

3 of riuT in the Betman°DK-2 Katio Recording S,,cetro|,,ioto,neler Tlie

Snleltrationotpltenantecncwascalcniatedfromta^^^^^

TSmoiar = ™ cyclohexaue at 252 m/i. and of DJIB.A from Js.|,„iiar

to test the efficiency of the analytical procedure, 2(1 //g. of
phenanthrene and 20 to 60 fig. of DilBA added to mouse skin were accounted for
to the extent of 98 and 99 per cent respectively.

DISCUSSIOX

The possibility existed that admixture of phenanthrene with DM BA might
reduce the absorption of the latter by the skin with a concomitant rcdtiction
in the tumour >deld. We observed, however, that the aijsorption of DM BA was
actually increased by mixing the two hydrocarbons in the ratios 1 : 4 and 1 : 2
respectively (Table I). It was onlj’’ when the ratio was 1 : 1 that the absorj)tion
of DjMBA Avas reduced.

The data of Table II show that the tumour jdelds induced by the mixliircs of
phenanthrene and DMBA Avere lower than the number obtained using DM BA
alone. This is particularly CAudent for the results showing that 1 - 21 //g. of DM B.\
per sq.cm, of skin in the absence of phenanthrene produced 2-2 tumours per mouse
Avhereas, slightly more DiNIBA (1-68 /ig.) in the presence of :b89 /ig. of ])hennn-
threne produced only 0-62 tumours per mouse. The difference is sicnificant at
P = 0-01.

It is unfortunate that the experiment Avas terminated at tlic end of tlio 20
Aveeks’ period of croton oil treatment. It is CA-ident from Table III that llie
production of new tumours in the group treated udth DJIBA alone liad sloppeti
but m the groups treated Avith mixtures of the tAvo hydrocarbons, new tuinouns
Avere stdl slowly appearing. Not only aa-us the rate of appearance of tiic tumours
slowed but the rates of growth seemed also to be slowed. None of tlie tumours
induced by the mixtures greAv quickly, or large, in sharp contrast to tJie bcliaA-iour
;apm“ phenanthrene. All the ttiourrA::;

that could haA'e been nrorbipprl nr « i. i number of tumouns

.piMce it is .ot possible to’say „„ thrp'S,o„S“

1-12 log (10 X ,,g. DlIBA) esSsS W SP’ = I'lS +

apply to the present exporimont (the ItaZfilt™ i^

594

TAX- YOUNG HUH AND J. A. McCABTER

Table II Distribution of Tumours Elicited by 2-5 per cent Croton Oil in Liqnid
Paraffin Applied Tioice Weekly to OFW Female Mice Treated Once on the
Back over an Ana of 1-54 to 1-66 sq.cm, with DMBA or Phenanthrene, or
Mixtuies^ of idle Two Hydrocarbons. Excess of the Applied Agent Washed Off
Skin Using Diethyl Ether 3 Hours after Application. Solvent for Hiidro-
carbons was Acetone. Interval between Initiating and Promotinq Treatment
was 3 Weeks.

Solution

Kumber

surviving

mice

Number of mice bearing following
number of tumours

Number

tumours

Tumours

Amount

hj’dro-

cnrbon

applied

Acetone

5 6 7 8 9 10

per

mouse

0-09

in skin
//g. sq. cm.

DMBA 0-2% . \

Phenanthrene — J

4 6 11

163

2*2

_ri-2i

DMBA 0-2% . \

Phenanthrene 0'05% j

— 2—1—1

1-47

/2-27
• \3-05

DMBA 0-2% . \

Phenanthrene 0-1% f

—

. 22

0-62

ri-cs

■ \3’3!)

DMBA 0-2% . \

Phenanthrene 0-2% J

0-97

/0-S7
• 13-32

DMBA — \

Phenanthrene 0-2% /

—

0-03

• {c-42

calculated that 0-62 tumours would be produced by 0-04 /(g. of DJIBA. This
result Avould impty that in this experiment, phenanthrene counteracted the initi-
ating activity of 97 per cent of the DMBA that was in the skin. Perhaps the
inhibition might have been complete had the phenanthrene persisted longer in
the skin, but the data of Table I show that it disappears at a somewhat faster
rate than DMBA. The latter substance might, tlrerefore have been free to act
though in small quantity and for a short time, in the absence of phenanthrene.

We are extending this work by applying the substances at different times in
relation to one another. A point of possible practical interest is that phenanthrene

Table III . — Latent Period of all Tumours Observed

Number of new tumours obsen-ed in following number of

^veeke

Solution

— — -

applied

DMBA 0-2% .

Phenantlirene —

. 3

DMBA 0-2% .

Phenantlirene 0-05%

. 2

(

DMBA 0-3% .

Phenanthrene 0 • 1 %

DMBA 0-2% .

. 1

Phenanthrene 0-2%
DMBA —

Phenanthrene 0-2%

. —

—

— •

—

PHENANTHREKE AS AN ANTI-lNlTIATING AOENI

is knomi to be a prominent constituent of tobacco tar (Campbell and Lindsey, H>oC.)
and tbe possibility exists that its presence there modifies the carcinogenic activity
of the tar.

This work was supported by a grant from the National Cancer Institute of
Canada.

SUMMABV

The absorption and persistence of 9,10-dimcthyl-l,2-bcn7,anthraccne (DM BA)
and phenanthrene in mouse skin were measured following apjdication of a mixture
of the two hydrocarbons.

MTien the proportions of the former to the latter were 4 : 1 or 2 ; 1 tlie amount
of DMBA entering the skin was greater than when DMBA was apjilied alone, but
the rate of appearance of tumours elicited bj* repeated treatment of the skin witli
croton oil was reduced. It is suggested that phenanthrene inhibit.s tlio initiating
activity of DMBA.

REFERENCE.S

Ball, J. K. axd McCarter, J. A.— (1960) Brit. J. Cancer, 14, 677.

Cajmpbell, j. M. axd Llxdsey, A. J.— (1936) Ibid ., 10, 649.

Crabtree, H. G.— (1946) Cancer Res., 6, 553.

HmL, W. T., Starger, D. W., Rizzo, A., Riegel, B., Shurik.

W. B.— (1951) Ibid., 11, 892.

Lacassagne, a., Buu-Hoi, axd Rudali, G.— (1945) Bril J exp Path 26 5
McCarter, J. A.— (1956) J. nat. Cancer Inst., 17, 399. " ’ ’ ‘

Idem, SzERB, J. C. abd Thombsos, G. E.— (1956) Ibid., 17 405.

^ Stanoer,

P. and WArtT.MAN.

BRITISH JOURNAL OF CANC15R

YOL. XIY

DECEMBEIL 1900

NO. ‘1

BETEL, TOBACCO, AND CANCER OF TIO-: :^I()UTn

G. S. iLUIR AND K. KIKK*

From, the Department of Pathology, Univcrrity of Malaya ni Siuifajyoir

Beceived for puWirntion Aupi-Jt 22, l!)nO

Tobacco has been, for many years, nndor snsj^icion as a carcinogen. Pride
oi place has oHate been accorded in the lifcratnrc to the reialioji.sljip between
bronclual caremoraa and cigarette smoking, but, in Sonlh Intiin and in tijose parts
of South-EaJ Asia where persons of South Indian stock work, or have .settled

^ problem of importance. F.sscntiidiv there arc twii
types of oral cancer; that associated with the chewing of tobacco nsualtv ad

.f ~ *'.c fon,...

complex chemistiy and pharmacology, re^M•e^YS tL^ c iiicartid ‘‘■'J

evidence pointing to the nresencp of s porn.-Jl • clinical and c.xpenmcntal
the quid, and reports expSents ^

Singapore betel quid. aqueous extract, of a typical

tlm Orient,:

Philypmes, blew Guinea, New Britain NW SnJ I»d«nc.sia, the

habit IS of great antiquity. The cbewhi^ Ireland, Formosa and China. qq,o

m the Sa„,fcrit ., S Jat/samhito >■ taK tXTi ” -I

mar B.nares, The Sanskrit for the iSTr S. f? -'.n (100

m the nodem Hindi “ tambnli anJin th, . v ' ”, PmH,

A.D. 1200-1400 ^ hnown to have reached tho 7^ derivation

on betel leafimperted fr ? I”"*' ®““>‘ arehives of fS r"

habit, ^ '»“1 gtowis, rafter thS to "T ''“'“'‘loo,

Tobacco is almost c "'•^"-ottnbtUml

598

C. S. MUIR AND R, KIRK

the leaf of tlie betel vine {Piper bale) on which catechu, an aqueous extract of
tlie heart wood of the acacme Acacia catechu or Acacia suma has been smeared,
bpices such as cardamom, cloves and aniseed may he added for additional flavour
banghvi Rao and Khanolkar, 1955). Tliis quid is inserted in the ginmvo-
buccal lold and chewed for hours (Shanta and Krishnamurthi, 1959).

In Thailand turmeric, the ground root of Curcuma aramatica is usually added
to the chew (Ellis, 1921). The aboriginal Veddas of Ceylon prepare tlieir slaked
hme from the shells of snails (Spittel, 1924), and coral is not infrequently used in
the Pacific Islands (Eisen, 1946).

IITiile the method of preparation in Malaya and Singapore is essentially
similar to that in India, the catechu is exhibited in a rather different form as
“ gambir ” {vide infra).

The betel vine is cultivated, the leaves used for chewing being on the hori-
zontal upper side branches ; the connoisseur’s leaf being the largest. Plucking
is done in the earl}’’ morning, and the leaves are protected from the sun to preserve
the aroma. The leaves are then bleached, the superior quality being very soft
and coloured a uniform green yellow (Burkill, 19356). This process heightens
flavour, which is due to the presence of volatile oils. Tlie cliief of these is eugenol,
an unsaturated aromatic phenol, usually very pale 5 "eIlow in colour, which has a
strong pungent odour reminiscent of cloves, and a pungent spicy taste. This
substance has antiseptic and local anaesthetic properties (Weatherb}^ and Haag,
1958). Chewing a betel leaf for five minutes leaves the mouth rather numb.

Terpenes are also present, these are pungent, and unpleasant if present to
excess. Unusually large amounts of potassium nitrate, and small quantities of
sugar, starch and tannin have been found (Mann and Patwardhan, 1916). The
chewed leaf is a gentle stimulant and carminative, sweetening the breath (Burkill,
19356).

The areca nut contains many alkaloids ; arecoline, arecaine, guvacine,
arecolidine, guvacoline, fso-guvacine and choline (Henry, 1949). Arecoline is the
only one of importance, the dried nut containing about 0-1 per cent. This
alkaloid is cholinergic, exerting a sialogogue and diaphoretic action in normal
dosage. Very large amounts depress the central nervous sj'’stein. It may e.xert
a deleterious effect on the dental enamel (Biker, 1 958). Also present are tannin,
the gljmerides of lauric, oleic and mju-istic acids, and a little sugar. This nut is
sometimes used internall 3 ' b 5 " the Malaj^s as a vermifuge and as a cure for diarrhoea

(Burkill, 1935a).

By itself the areca nut is highly acid and astringent to the taste. The addition
of lime not onlj" neutralises tliis to a large extent, as can easily be demonstrated
in vitro, but also promotes the appearance of a red d}^.

From the shrub Uncaria gambir the “ getah gambir ” of the Malay, the ’ katta
kambu ” of the Tamil, is extracted. The dehcacy of flavour of this product
depends upon its oatechin content. The leaves are bound, steamed, and then
small amounts of boiling water are allowed to trickle through. On cooling
catechin crv-stalhses out, leaving the more soluble, and bitter, catechu tannic acia
in solution." Usually a little bran is added and the bran-catechin mixture made
into cakes. In the production of gambier for tamiing a crude process is used,
extracting most of the catechu tannic acid (Burkill, 1936c).

Apart then from the tobacco nicotine which may be present in the quid, an
which apparently has the same power to effect habituation as that from smoke

BETEL. TOBACCO ANW MOUTH CAXCEU

r.!)!)

tobacco the betel leaf-areca uut-gambir-limc mixture promotc.s "

fFisen ’l946) mild exliilaration, and to a certain extent, siceple.ssiic. I i-
Sential oUs ’give a pleasurable tang and impart a subjective confide.i^ce m tilt
the ^vholesomeness of the exhaled breath. However, like other habit s of a similar
nature, betel chewing seems to he an acquired taste, the first chew, uliet her
tobacco is present or not, causing giddiness and nausea. It slioiilcl also he no ed
that the colour red, such a prominent feature of the chewed quid, and of t i
expectorated mouth juices, connotes good luck to Indians, Chmc.so and rilala.vs.
as well as to other Asian peoples. Betel chewing is a poor man s luxury, as a
made-up betel/tobacco quid costs 5 cents local currency (one penny sterling) ; t Ins
is about the price of a cigarette which lasts for a much shorter time.

Tobacco is smoked in many parts of India in the form of “ hidis , a variety of
cigarette in which dried powdered sun-dried tobacco (Xicoliniin inbneum or
Nicofiana rusticum) is wrapped in a variety of leaves, usually the dried leaf of
the tembumi (Diospyros melanoxylon), the whole being secured at one end by a
thin string (Sanghvi el ah, 1955). These “ bidis ” are not generally smoked by
the Chinese or Malays in Jlalaya or Singapore, but they are popular with locally
domiciled Malayalees and Tamils of Social Class V. A similar type of smoke is
verj' popular uith the Mala3% the “ rokok daun ” compo.scd of a thin central core
of Siamese tobacco around which is uTapped the leaf of the Xijiah jialm (Xipnfi
fruticans).

The taxonomy of the plants mentioned above has been described by Bidlev
(1922-25).

Clinical Evidence

The main interest in betel chening has, of course, always been directed to
its possible relationship uith oral cancer. Many of the early writers hold there
was no such relationship (Maxwell, 1924; Wells, 1925), whereas others (Fells.
1908 ; Bentall, 1908) did. Davidson (1923) and Spittel (1924) pointed out tlie
possible sigmficance of tobacco in the quid. Most of the confusion arose from a
failure to distin^sh between betel and betel/tobacco quids. Ellis (1921) held
a survej' of medical opinion in Siam, the consensus of which was that there was

the matter further (Mendelson and
Ellis, 1924), he showed that m the 24,340 males attending Government clinics in
Bangkok in 1922--23 there were 43 cancers, 49 per cent of which were in the
mouth, and concluded that betel/tobacco was carcinogenic. Others such as

S: ^agenf ™ that iSi’e uns tlm

CIiiiica.1 evidence has slowly accumulatpfl nvpr tTid Troo™
tions tending to be more accurate statistically. ’ ^ mvestiga-

Orr (1933 in his classical description of oral cancer in EpI-pI /f^i i
examined m detail 100 cases of oral cancer. Of these 2 were
chewed occasionally, 24 chewed from tE,-pp ^ chewers, 9

than this number, and 25 slept uath a ouid in ^ ’ shewed more

corresponding figures wre 34

does not state how his control group was TTp ’ f ’ / ? “•

thirds of the cancers involved the site directlv imT^f shoned that over two-
inoiirs were on the lower alveolus, or between Thf^f'

’ »«’^"een the alveolus and the cheek.

600

C. S. MUIB AND E. KIEK

33 per cent involved the cheek alone, 15 per cent the tongue and the floor of the
mouth, 10 per cent the upper jaw and palate, and 8 per cent the lips. He re-
marked that the lip tumours seemed to begin just inside the angle of the mouth
and wondered if this might be due to the liabit of squirting juice out of the comer
of the mouth.

Eisen (1946) described betel chewing in the Southwest Pacific Islands. Here,
as well as the areca nut, the leaves and pods of the piper belle are used. The
lime is obtained from sea-shells or from coral. Tobacco is not used, and cancer
of the mouth is virtually unknown ; one case in 8000 adults admitted to a Heir
Guinea hospital. The teeth of these chewers, although stained red, were in good
condition, unlike those of the betel/tobacco chewers of Madras (Slianta and
Krislmamurthi, 1959).

That this susceptibility to oral cancer follmving the chewing of tobacco is
environmental rather than racial is adduced from the evidence of Priedell and
Rosenthal (1941), who described 8 cases of oral cancer in wliite American males
who habitually chewed tobacco per se. These tumours arose at the point where
the quid was usuallj'- placed.

Khanolkar and Sur 3 'abai (1945) describe an unusual cancer of the lip asso-
ciated vdth the use of unsmoked tobacco. This thej' found particular^ prevalent
in Bihar, in north-east India, accounting for a minimum of 12-6 per cent of oral
carcinomata biopsied. They noted that patients u'ith this cancer sought treat-
ment at such a late date that biopsj’’ was often considered unnecessarjf. This
undue prevalence they considered was due to the use of “ khaini ”, a powdered
adnuxture of dried tobacco leaf and lime. A pinch of the mixture is deposited
in the groove between the front lip and the teeth, being left there, until after
dilution by saliva, it is swallowed. This process is carried out at frequent inten’als
tluroughout the daj’".

Sanghvi et al. (1955) undertook a statistical surve}’' of 1460 patients referred
to the Tata Memorial Hospital, Bombay, in 1952-54:. Patients referred to this
hospital in whom no cancer was detected formed a control group. Patients were
asked whether thej’' smoked “ bidis ” or chewed betel. It was shomi that chewing
of betel /tobacco was associated with cancer of the oral cavity ; cherving and
smoldng with tumours of the hypopharjmx and base of the tongue; smoking
alone with cancers of the oropharynx, notably the tonsil, and the oesophagus.

Shanta and Krislmamurthi (1959) reviewed the 347 oral cancers seen in one
jmar at the Cancer Institute, Madras. 71 per cent of aU oral cancers (26'45 per
cent of all malignancies) arose from the buccal mucosa, and 22 per cent (8 per cent
cent of aU malignancies) from the lingual mucosa. The incidence of cheek cancer
w'as higher in the male ; environment, religion, anaemia, sjqjliih's, tuberculosis,
chabetes, hjqjertension, virus diseases and achlorhj’’dria were not of si^fieance.
The habit of chewing tobacco, betel leaf and areca nut was higlily significant.
85 per cent of those with a buccal cancer chewed all three, wdiile in the non-
cancerous control group the figme was 12-5 per cent. Only 8-7 per cent, of those
with a cancer chewed betel nut and lime alone ; of the control group, 51-8 per
cent. Smoking did not appear to be of importance, but gross dental sepsis was
considered to be the main factor in the Iiigher incidence of these tumours in the
labouring, when compared with the lower middle class.

In carcinomas of the anterior two-thirds of the tongue the same factors hela
good, although tobacco smoking appeared to pla 3 ’^ a dominant role in cancers o

BETEL. TOBACCO AND MOUTH CANCI'.K

()l) I

4.1 f Sindivie/rt? (1955) attributed the higli incidence of pastorior

Another tvue of cancer associated with smoking is tl at of he harrl l_u ate.
Tliifis for the^most part confined to Vizagapatam, and tlic outlying ^

AiXa Province, Xch is situated in the mid-eastern jiart « «

cancer is almost certainly due to the smoking of chutta , a t'.vpo ^ig
S by rolling dried tobacco leaf, which is then tied at Uic end w.tli a Inn ijic c
of striS (lOianolkar and SuiTabai, 1945). Such a primitive device is difiiciilt to
smoke as the smoke is not easy to draw, the core being vcp' nrcgulai. .'Vs th .
lighted end goes out easily it is customary to keep it inside tlic niouth to inomote
combustion Por some reason it is onlj' the women who use this adda jioga ,
or reverse smoking, men from the same district, and of the same social standing,
smoking the chutta in the orthodox manner (Reddy, Reddy and Rao, HHid).

Experimental Evidence

Woelfel, Spies and Cline (1941) tested the ether, alcohol and unsaponifiable
fractions of areca nuts on mice, but failed to evoke tumours. Wliilc prolonged
subcutaneous injection of tannic acid (tannin, gallotannin) has been shown to
produce liver cirrhosis and eventually liver neoplasms, this substance did not
evoke local tumours (Korpassy and Mosonjd, 1950). Although tannin is present
in areca nut, and both catechu and catechu tamiic acid may both be classed as
tannins, their chemistrj’’ is so complex (Nierenstein, 1948) that each substance
would need to be tested for carcinogenicity. To the best of the authors’ belief
no work has been done on the other constituents of the betel quid ajiart from
tobacco.

Roffo (1939a, 19396) prepared several distillates of tobacco ; a watery extract
(100 to 120° C.), a thicker liquid (120 to 350° C.), and the residue. These'products
were applied daily to the ears of three batches of 20 rabbits for a period of 10
months. The first distillate evoked no tumours, but 95 per cent of those painted
with the second, and 70 per cent of those painted with the residue developed
squamous carcinomata. Sugiura (1940) was unable to confirm these findings
using comparable distillates. Roffo (1941) showed that nicotine alone was not a
carcinogen for rabbits.

Extracts of sun-cured Indian chewing tobacco have been prepared by Mody
and Ranachve (19o9) and assessed for carcinogenicity by painting the intcr-
scapular skm and buccal mucosa of Strong (A) and S^’ss mice. Some ex racts

M.., the « L„th „t

used on the buccal mucosa, udthout apparent result TLp incp f
quids into the cheek pouches of hamstS proved Effective 3 4 r '”
pyrene used as a control carcinogen produced cancerTpn i • J ^
cliemical placed on the buccal mucosa had no effect the ^

Le^^^ Gorlin, and Gottsegen (1951) that the abIp r authors suggesting, like
«s a portal of entry, and the 81“"* ^ act

vanced hjT,erpl,stic changes ">"cm were rsponsible. Ad-

alk,aloid eontaining extracts to the skin when stanh“ “f

, wnen simultaneously painted with

602

C. S. MUIR AND E. KIRK

croton oil, but a poor survival time did not permit further study. The effect of a
single subcutaneous injection of extracts was also followed. One mouse, injected
with a total extract, developed a transplantable palpable fibrosarcoma in the
subcutis not far from the site of administration. Large numbers of apparently
spontaneous tumours were seen in the animals used in these experiments, but they
occurred with equal frequency in the control stock.

Johnstone and Plimmer (1959) in their exhaustive review of the chemical
constituents of tobacco remark, “ a large amount of research has been concentrated
on the aromatic hj'^drocarbon content of tobacco smoke, whilst fresh and processed
tobaccos harm attracted specifically less attention ”. hfevertheless 3 : 4 benzo-
pjT-ene has been identified in extracts of fresh and processed leaves (Bentley and
Burgan, 1958). There is some evidence to suggest that this may be derived from
the atmosphere (Campbell and Lindse 3 q 1956).

There is of course ample evidence to show that the tars produced by pjTolj^sis
of tobacco contain carcinogens (Wynder, Graham, and Croninger, 1953). Beddy
et al. (1960) describe experiments in wlrich the effect of tobacco tar, produced by
burning “ chuttas ” and dram'ng the smoke through acetone, was determined on
mice. Painting of the backs of the mice with the tar alone, basal cell proliferation
and hjqierplasia of the sumat and sebaceous glands were seen. There was no
evidence of anj^ neoplastic change at the end of four months. When heat was
applied to the skin of the mouse after painting with tar, earty malignancy was
seen bj'- the third month, and invasion of the dermis by the fourth. A tempera-
ture of 58® C. was chosen as it had been shown, by thermocouple, that flu's was
the palate temperature of the “ chutta ” smoker. Iflianolkar and Siirj'abai
(1945) using a long stem thermometer recorded a somewhat liigher mean tem-
perature of 65° 0. Beddj'’ et al. ( 1 960) concluded that there was not onl}’’ a shorten-
ing of the latent period when heat was used, but there was an increased tumour
yield, and felt that these observations explained the lugher incidence of cancer
of the hard palate among “ chutta ” smoking women.

In view’" of the comparative paucitj'^ of published work, and that conflicting,
it was decided to paint the ears of Swiss -white mice -with an extract as near to
that present in the mouth of the betel/tobacco chewer as could be devised.

The paint was prepared daily’- just before use. Tlxree betel vine leaves were
placed on a bench, and the inner smeared -svith moist stone lime. The equivalent
weight of dried lime was about 0-2 g. On this were placed shavings of bete
nut, approximately'- 4-0 g., and about 0-5 g. of “gambir” together -with aboiv
1-0 g. of sun-dried tobacco imported from South India. (Indonesian tobaccos
are sometimes used in Singapore.)

The leaves were then -UTapped round these ingredients and the whole trans
ferred to a brass mortar and pestle to be ground for 5 minutes. 2 ml. of
were added, mixed throughly with the ground material, and the resultant dsuK
red mass squeezed hy the fingers. The fluid so obtained, temperature -
also dark red in colour, was then painted on the ears of the mice by^ a mm er o
camel hair brush and allowed to dry. Drying took about 10 minutes. '

first few days the mice seemed rather irritated, but thereafter the act of > g
did not seem to worry them, nor did they make attempts to clean e e
* *c© ofiP

^'^^^Lflially a pilot trial using 12 numbered Swiss white, brother-sister mated,
mice was instituted. They were painted daily for two years. Special care w -

BETEL. TOBACCO AKD MOUTH CANCEB

r.HH

A ill till*

taken to ensure tltat the mice hn;! nn „nse

StS4ior„f||S’t‘i: tai “nto^m"

=SS:S=l==ss;i:r.ri.,

noted^rbrSQ w«.H Htoppjvl

atd tli ear biopsied.’ After healing, the wound broke

later. Ulceration of the riglit ear occurred one inontli after the left » '

revealed loss of a large part of the ear, the bare area being covered bj a fil n
cap which contained moderate numbers of polymorphs. The remaining sKii
towards the base of the ear, on both inner and outer aspects, showed BBirkw
thickening, often focal in nature and numerous intra-cpidernml keratin tilled
cysts. There was no evidence of malignancy and inflammatory change.s were not
prominent.

Similar changes were seen in a second mouse at much the .‘;amo time, and in
a third, one month after painting had ceased. Ulceration of the left, car cont iiuied
slowly over three months, when the right ear showed similar changes.

A fourth mouse showed ulceration of the base of the right car laterally :12
months after the trial started, i.e., 8 months after painting had slopped (Fig. I ).
A regional lymph node was markedly enlarged on palpation.

Sections showed an invasive squamous carcinoma (Fig. 2) arising in a polyjwid
excrescence containing numerous keratin filled cysts, some of which communicated
with the surface. Cell nests were prominent. Parts of the invading tumour had
reached the perichondrium. The skin on either side of the tumour was (hickeiicd
and showed marked appendage change. The lymph node was invaded by a well
differentiated squamous carcinoma of the same general structure as llic priinarv
tumour (Fig. 3). There was some doubt as to whether this was a direct extension
or a metastatic phenomenon as the lymph node was somewhat torn. 'J’he sub-
capsular sinus showed marked catarrh.

The other ear showed thickening of the epidermis and ulceration of the tip.

In a fifth mouse, six months after painting ceased, a large subcutaneous
nodule was seen to grow rapidly, just to the right of the midlinc, some l-.o cm
behind the ears, reaching in five weeks a diameter of 1 -5 cm. The swelling became
ulcerated and the mouse was killed. The tumour was almost globular in shape
and had a weh marked pseudo-capsule, which was significantly infiltrated with
round cells. Histologically a basi-squamous carcinoma, there were, principally
at the growing margin, numerous islets of dark staining basal cells intermimrIpH
with very small differentiated cell nests (Fig. 4). Largf areas at tL ppT ^
mummified, Mected and necrotic. Calcification was sLTsetal pS^ 'Z

of which showed signal nuclear at^ia. SimSa? bu/£^^
normal were present in most of the contimimio « • i’ • ^<3partures from

In a sirfh mouse a S™ sQ„ar^?^ril? ™ “ "> ''“‘I' n"™-

ear and the rear fold of SorfCr'S' w.T ^n® found between the right

on either side showed moderr Skei^Td T” The skin

(lea-th, of t.IiA ^ 9'ncl somo aiBOGriflawA ^

. of tke mmatata, s. mice,

_e. At
subcutaneous

604

C. S. MUIR AND B. KIRK

sepsis at the root of the ears, in two there was no demonstrable lesion in the fifth
there was a reticulum type sarcoma. The sixth was discarded in error.

Painting of a second batch of 41 mice was begun in April, 1958, and is still
in progress. The first lesion, noted six months later, was a papilloma situated
at the base of the left ear. It was removed surgically. The epithehal covering
Avas thick, ^fferentiated and hyperkeratotic, being considerably infolded to form
large keratin filled cysts (Fig. 5). There was one small area of questionable
break-through. The subjacent dermis was rather oedematous and was conspicu-
ously infiltrated by acute and chronic inflammatory cells. Numerous small
blood A'essels ivere noted.

The second lesion appeared in another mouse 7 months later. The left ear
began to ulcerate, and ivithin 15 days the entire ear had vanished. Painting
was stopped as soon as ulceration was noted and CAmntually the ear site healed,
to break doum again six months later udien it rapidly extended on to the neck
tissues. The mouse Avas killed. Although infection, ulceration, and extensii’e
epidermal and appendage changes AA'cre noted there was no focus of unequivocal
malignancy. Gross thickem’ng of the interscapular skin was seen in one mouse,
and minor degrees of ear ulceration in two others.

Li all a further 10 mice in this group haA'e died. One had multiple tail and
lung abscesses, another a blood-stained ascites of unknoAvn cause, a third a form
of gross ataxia, again of unknoum cause, a fourth a liver cell carcinoma. No
obAdous disease urns seen in the remaining six.

EXPLANATION OF PLATES

Fig. 1. — Mouse ear showing malignant ulceration. This could not be distinguished by the
naked eye from non-malignant ulceration. The black pigment at the ear tip is dried paint.

Fig. 2. — Part of a well differentiated invasive squamous epithelioma from the mouse in Fig.
I . Note the large number of keratin-fiUed cysts on eitlier side of this portion of the tumour.
H. & E. X 27.

Fig. 3. — Well differentiated socondarv tumour gi-owth in lymph node. Same mouse as Fig. I
and 2. H. & E. X 90.

Fig. 4. — Basi-squamous carcinoma with clumps of basal tj-pe cells at the centre and cell-nest
structures at the periphery. H. & E. x 90.

Fig. 5. — Squamous papilloma with a large keratin filled c>-st. H. & E. x 18.

Fig. 6. — Section of normal mouse ear skin showing relatively undifferentiated two-layer
epidermis. H. & E. X 370.

Fig. 7. — Stouse skin showing marked increase in thickness and in numbers of cell strata, with
differentiation into basal, prickle cell, granular and keratin layers. H. & E. x 370.

Fig. 8. — Numerous small intra-epidermal cysts, filled with keratin, in hj-perplastic skin at tlic
base of an ear. The panniculus camosus is seen at the bottom right. H. & E. X HO.

Fig. 9. — Grossly thickened and hyperplastic mouse skin. Note the large prickle cells.
Mitoses are not prominent in this particular field. H. & E. X 370.

Fig. 10. — ^An epidermal process composed of well differentiated prickle cells, in close contact
ivith, if not actually' invading, the aural cartilage. H. & E. X 873. ^ ,i- ,

Fig. II. — ^The hjiierplastic hyperkeratotic epithelium shows grossly shortened hair follicles
whose mouths are filled by keratin plugs. H. & E. X 100. . r „■ i rl

Fig. 12. — ^Altered epidermal appendages showing remnants of botli hair follicles (a) ana
sebaceous gland structures (b). A clump of basal cells is stUI present (c). Epidermal
thickening and hj-perplasia is obvious, H. & E. X 100.

Fig. 13.— Large hyperchromatic appendages which have undergone complete squamous
metaplasia. Under higher magnification the basement membrane of the arrowed toincie
seems to be absent. Such appendages may be difficult to distinguish from enlarged epi-
dermal downgrowths. H. & E. X 100. . , ,

Fig. 14. — Cartilaginous metaplasia in fibrous tbsue. The metaplastic cartilage is
human type, not of the simple murine seen below. H. & E, x 100.

Fig. 15. — The hair follicles are shrunken into small sub-epithehal clubs or wedges,
infected keratin cyst is seen on the right. H. & E. x 100.

BltlXISII JoUKN’AI. OK C'AN('|;I!.

Vol. MV \n. .|.

Muii’ ,„)d Kiik.

BrITTSII .loiTlN'AI. OF CA-NTFI!

Vol. XIV. Xo. (

■'''"irnnd Kirk

BETEL. TOBACCO AND MOUTH CANC;EU

fior

The ears and surrounding skin of for' nnnndiiT

feed intervals, nevertheless by study of the ^v^dablc sections « f url> com,

In regions udth a reduced number -

tliicker and layermg more obvious (Fig. 6) but nowhere docs it. njiiiroaeli tlu

human type of epidermal covering. , . „

The earliest change occurred in broad foci. Tlie basal cells came to occu,i\
the entire basement membrane : a concomitant increase in the niiinber of s|)iiious
cells gave rise to a definite priclde cell layer which differentiated into sr/.cable
granular and keratin strata (Fig. 7). Irregular maturation of the Icerat in layer
was seen, characterised by the presence of large cuboidal nucleated cells with an
intensely eosinophilic cytoplasm. Elsewhere keratin was present to excess, often
coated with particles of pamt. Small intra-epidermal keratin-filled cysts were not
uncommon (Fig. 8).

As well as this change into the human tj^pe of epidermis, there were focal
areas of yet further hj^ierplasia. Here the prickle cell layer was nmch thicker,
the spinous cells larger, the basal cells stained more deeply, and mitoses were
present in increased numbers (Fig. 9). From such areas, epidermal domigrowths,
often apparently without basement membrane, penetrated for varying distances
into the dermis. These do-wngrowths remained very ■well difTerentiatcd, and were
considered benign, although one such w'as seen to be in close contact, with, if not
actually invading the aural cartilage (Fig. 10).

Conspicuous epidermal appendage change was seen in about 05 ])cr cent, of
the mice. Hair follicles seemed shortened, their mouths filled by keratin plugs
(Fig. 11). Sebaceous glands showed squamous metaplasia, many retaining a
thin basal cell layer on the outside (Fig. 12), while in a few this 'seemed to bo
absent (Fig. 13). Many of the appendages were scarcely recogni.sablc as .such
and were it not for the persistence of a central structure derived from the lumen’
of the original hair follicle, containing a small piece of keratin (Fig. 12) or flic
presence of clumps of large granular sebaceous cells (Fig. 12), would have been
indistingurshable from enlarged epidermal domigrowths (Fig. 13).

TOceration was common, invariably begimiing in areas of hyperplastic eni-

STaranermJs orfih forniatiL

mth parallel rons of fibrocytes beneath an intact epithelium heinn a fm

quent finding. In one mouse cartUaginous metaphasia was induppfl^ni
connective tissue laid down close to eti- induced m new

a human rather than murine type (Fig 14 ^ P^ eilSnJ colf'^®''
normal. i v ^ J"re existing collagen appeared

the areas ot epidetilal'th!L'eiiim''aSwe^a''''^ many, but not nil, of

tbo epWemis bordering on thfmalignant tnmonre’" ''“'MUi

iTgrr "

606

C. S. MUIR AND R, KIRK

In the contro] mice there were two spontaneous malignancies, one hepatic
tlie otiier probably uterine or ovarian, this latter ulcerating through the hinci

DISCUSSION

It is difficult to evaluate how much of the change in the ears and surrounding
skin was due to irritation by non-carcinogens, and how much to the carcino<ren
itself. Orr (1938) painted mice with benzene, a moderately powerful irritant, and
observed, Aidthiii 6 Aveeks, epithelial h 5 rperp]asia and some depilation. ' The
h 3 'perp]asia AA'as of the same nature as that seen with knoini carcinogens, but the
number of cell layers AA'as not so great. Man3^ of the hair follicles Avere enlarged
and hyperplastic Aidth swollen internal root sheaths and enlarged hypercliromatic
bulbs. There atos never the appearance, such as is seen after one week of ni 0 th 3 d'
cliolaiithrene painting, Avhere almost every hair follicle is slmunlcen into a small
sub-epithelial solid AA^edge. This latter picture was not infrequently seen in our
mice (Fig. 15), and indeed, most of the changes in our animals, if obserA'ed in a
mouse painted AAuth a knoAim carcinogen, would be labelled as preraalignant.

Dermal changes are equall}'^ difficult to assess. Gross infection Avas common.
In mice with earl 3 '' epidermal changes, no alteration AA'as seen in the collagen
comparable to that described b 5 ’^ Orr (1938) and others. Perhaps this may be
ascribed to the relative AA'eakness of the carcinogen.

The aAmilable clinical and statistical CAudence all points to tobacco as the
carcinogen in the betel /tobacco quid. Why our relativelj' crude e.vperiments
should haA'^e apparentl 3 ’’ succeeded in producing malignancies when the more
sophisticated techniques of Mod 3 ’' and Ranadive (1959), using purer and stronger
tobacco extracts, have failed, points, we feel, to the importance of co-carcinogenic
factors. Wliich of the many components of the quid is of the greatest importance
remains to be discoA'ered, or, indeed, it may be that tobacco is the co-carcinogen
for some other substance.

The arbitrar 3 '’ choice of the ear for painting ma 3 '^ haAm been responsible in
some measure for our success. Here the amount of underlying dermis is small,
and is backed b 3 ' a plate of relatively avasoular cartilage. An}^ carcinogcAA
passing tlrrough the epidermis atos likelj'^ to remain there for some time before
absorption, and not diffuse through to deeper tissues. The products of dermal
degeneration, whether caused b 3 ' irritation, or by the carcinogen, are also more
likely to promote a neAA'growth in the oA'erlying dermis, as has been suggested b}'
Orr (1938) and Marchant and Orr (1953). In tliis connection, it is noteAVorth 3 '
that although the tongue is as near the heat and smoke from a “ chutta ’ smoke^
AA'ith the burning end in the mouth, as is the hard palate, Beddy and Eao
in a series of 107 oral cancers in female smokers, found 68 palatal cancers for 14
lingual, i.e., 5 to 1. The dermis of the tongue has eas}’’ access to the uiidertying
tissues ; that of the palate is backed by bone. Similar conditions obtain in le
iimer aspect of the labio-gingival fold (“kliaini ” cancers) and in the inner aspec
of the buccal sulcus, but not, of course, on the cheek itself.

Although at present, as Avill be eAudent from the papers quoted, a pro cm o
magnitude, it is our belief that the incidence of betel chewers cancer will lai .
particularly in multiracial Singapore, as liA'ing and, probably
educational standards improAm. In Malaj^a this decrease AA'ill be nmc i s ‘
the isolation of South Indian labour on rubber estates and on ru ic

betel, tobacco AXl) MOUTH CANCl'Hl

(KtT

Department gangs, is responsible for the iKrsistence of tl.e olKoving bnl.it. ns a
closed community gives up such customs less rcadilj .

SUMMARY

The geographical distribution, the history, and sociological iiuimrtancc. of

betel chewing are briefly mentioned. .

The chemistry and pharmacologj' of the contents of the (jnid arc reviewed.
The clinical, statistical, and experimental evidence pointing to tobacco a.s the
carcinogen in the chew is examined. The failure of early writers to mention
whether tobacco u'as present in the quid chewed is shoum to have led to some

confusion. ^ . i - •

Experinicnts are described in "whicb the cars of 5.^ Swiss wnite mice ^^^'r(*
painted daily for two years with an aqueous extract of a typical .Singapore betel /
tobacco quid.

In the first batch of twelve mice, all of which aic now dead, two siiuamoiis cell
carcinomas and a benign squamous papilloma appeared on or around the painted
area, either during painting or after it had ceased. In a second batch of 41
similar mice, 30 of which are still alive, one active squamous pajulloma has been
noted to date.

The vaiying degrees of epidermal and dermal change seen in the cars of all the
mice are recorded, and are found to be substantial I}" the same in appearance as
those caused by the carcinogenic cjmlic hydrocarbons.

We wish to thank Professor J. W. Orr for useful advice and histological ojiinion.
Mr. V. Nalpon for the photographs, our technical staff for the many sections.
Mr. Rengasamy for care of the animals, and Mr. P. A. Samuel who’ typed tin*
script.

REFERENCES

Bextall, W. G.— (1908) Bril. med. J., ii, 1428.

Bextley, H. R. axd Burgax, J. G.— (1958) Anahjsl, 83, 442.

BuRKHmL, L. H.— (1935a) ‘ A Dictionary of the Economic Products of the idalnv
Peninsula , London (Gronm Agents for the Colonies), Vol. I, n. 223 — (*HI3r?M
/bid., t ol. n, p. 1/3/. — (1935c) ibid., Vol. II n 2198 1 •• )

Gajipbele, j. M. axd Lixdsev, a. J.— (1956) Bril. J. Cancer 10 049
Da%tdsox, j.— (1923) Bril. med. J., ii 733. ’ ’ '

DA^^s, G. G.— (1915) J. Amer. med. Ass., 64, 711
Eisex, M. J.-(1946) Cancer Res., 6, 139.

Ellis, A. G.— (1921) Arch, intern. Med., 28 252.

Iells, a.— (1908) Brit. med. J., i, 1357

.roimsiiisE^E, i tv! aS iinnta'j'p''* (CImrelrill), p. s.

V^axolkar, V. R. Axn .Suryab™ B f 885-

Korpa-ssy, B. axd Mosoxyi, M -(lobSf fn'
hKvv. B. M., Gorltx. R. axd GomKCEN r

Aaxx, axd Patu'ardhax — (19161 ilfp-m n Cancer Inst 12 ajr,

burkhm (10356). W., 4, 32)! ' ftmltd i,v

608

C. S. MUIE AND R. KIRK

aiAECHAKT, J. AND Ork, J. W— (1953) Brit. J. Cancer, 7, 329.
jMaxwell, j. L. — (1924) Brit. med. J., i, 729.

McOaekison, R. — (1931) Ibid., i, 966.

Mendelson, R. W. and Eelis, A. G.— (1924) J. trap. Med. [Htjg.), 27, 274.

Mode, J. K. and Ranadev^e, K. J.— (1959) Indian J. med. Sci., 13, 1023.
Nierenstein, M. — (1948) in Allen’s Commercial Organic Analysis. 5th Ed. Phila-
delphia (Blakiston), p. 24.

Ore-, I. M.— (1933) Lancet, ii, 575.

Orr, j. W. — (1938) J. Path. Bact., 46, 495.

Reddy, D. G. and Rao, V. K. — (1957) Indian J. med. Sci., 11, 791.

Idem, Reddy, D. B. and Rao, P. R. — (1960) Cancer, 13, 263.

Ridley, H. E. — (1922-25) ‘ The Flora of the Malay Peninsula ’. (5 Vols.) London
(Reeve),

Riker, W. F., Jr. — (1958) in ‘ Pharmacology in Medicine ’ Edited by Drill, V. A.,
2nd Edn. New York (McGraw-Hill), p. 367.

Roffo, a. H.— (1939a) Dlsch. med. Wschr., 63, 1267.— (19396) Ibid., 65, 963.— (1941)
Schweiz med. Wschr., 71, 549.

SANGim, L. D., Rao, If. C. M. and ILhanolkar, V. R. — (1955) Brit. med. J., i, 1111.
Shanta, V. AND IfRiSHNAMURTHi, S. — (1959) Brit. J. Cancer, 13, 381.

Spitted, R. L. — (1924) Brit. med. J., i, 158.

SUGIUBA, K, — (1940) Amer. J. Cancer, 38, 41.

SnNTZEFF, V., CoivDRY, E. V. AND Groninger, A. — (1955) Cancer Bes., 15, 637,
Weatherby, j. H. and Haag, H. B. — (1958) in ‘ Pharmacology in Medicine ’. Edited
by Drill, V. A., 2nd Ed. New York (McGraw-Hill), p. 108.

Wells, C. R.— (1925) U.S. nav. med. Bull, 22, 437.

WoELFEL, W. C., Spies, J. W. and Cline, J. K. — (1941) Cancer Bes., 1, 748.
Wynder, E. L., Graham, A. E. and Groninger, A. B.— (1953) Ibid., 13, 855.

histological types W a LUNO CANCEK MATEUIAL

liS VrjiNiOJlj

E EERRARI akd L. KREYBERG

Received for publication September :

, liinn

Lv a series of countries a neu* lung cancer sttuation has been untlcr
development in the present century. The new situat.on ts 1 h> n

increased occurrence of lung cancer, especially m males in urban areas. I he
increase is mainly caused by carcinomas histologically desigiiatcfl as cp.dcniioitl
(squamous cell) and small-cell anaplastic (“oat”-ccll) carcinomas, whereas (he
other types of primary epithelial lung tumours seem to show a more coiistaiit
and equal occurrence in males and females, in urban and rural areas, in all larger
occupational categories as well as in smokers and non-smokers (Ivrcjbcrg, Ih.i.l).

Even if increased cigarette consumption is accepted as a major cause of this
new situation, other factors, suspected or unsuspected, arc ]irobably involved niifl
an international sun-ey of the lung cancer development in different counlries.
with different habits and living conditions, is highly desirable, and the M'orld
Health Organization in its Technical Report Ho. 192 (1900) has recommended
that such studies be made.

As part of such a programme the Director of the WHO International Reference
Centre for Histological Definition of Lung Tumours (Professor L. Kreyberg)
approached the Director of the pathological sendee of the Ospedali Civili Rinniti
in Venice (Professor E. Ferrari) with a view to obtaining a preliminary orientation
from a site exhibiting some peculiar conditions.

Venice is a town without automobiles. Tar and asphalt arc used as street
covers in negligible amounts, heavy industrj^ is absent and no coal smoke polintc.s
the air.

On the other hand, Bastai and Pescetti (1959) have confirmed tlie statement
of Fabris (1937) that in Venice lung cancer is just as prevalent, if not more .^o as
in other Italian cities. ’

Ferrari has from his autopsy material found that in males two out of evcr\-
seven cases of malignant tumours are lung cancers, and that lung cancer is twice
as frequent as stomach carcinoma, the second in numerical importance

in the Vemce autopsy material the sex ratio for lung cancer as a mean of f l.n

m^ps^nmteriaVis^“^^^^^^ corresponding sex ratio for ‘the total

"■ '-onioc i.

.1.0

ratio uroup i tumours fenidprmmVi u uivsis mat tlio

cinemas) to G„,.p ’‘"1?';=“'; <“ 0»0'

carcinoids (“ adenomas ”) and mLous glanTr ^"‘rcinomas,

indicator of the development of the new luno- na g| 2 nd ) tumours) is an

could be used as a meLure of -tio

cancer m areas where

610

E. FERRARI ANB L. KREYBERG

incomplete mortality and morbidity statistics only are available and it was
proposed to plan an international geographical study of lung cancer on this basis

Ihe World Health Organization has accepted these premises as a vorkina
hypothesis. «

A study of this special problem therefore was included in our survey.

For the period 1956 to 1960 Ferrari was able to collect 78 cases for liisto-
logical typing, and this tj^ping has now been completed according to the nomen-
clature recommended for trial by an Expert Committee under the auspices of
WHO.

The result of the typing is shown in Table I.

Table I. — Distribution of Histological Types in Venice

Tj-pe

Group 1

Group II

Others

F V

, « ,

, ^ ^

Sex

Total

Males

Femnle.s

. 7

Total .

Ratio Group I : Group II.
Males 45 : 14 3'2:1.

E Epidermoid carcinoma.

O Small (“ oat ”) cell carcinoma.

A Adenocarcinoma.

B Broohiolo-alveolar carcinoma.

C Carcinoids.

U Anaplastic and uncertain tj’pes.

M Combined epidermoid adenocarcinoma.

Even if the material is limited it shows some remarkable features.

The first is a ratio Group I: Group II tumours of 3'2:1 in males. The cor-
responding ratio for Norwa}'’, a country ■noth a known low incidence, is 3-4:1, as
the mean for the last 12 years. According to the thesis of Krej'^berg tliis vould
indicate a rather low total incidence of lung cancer also in Venice, a conclusion
at evident variance with the frequency figures quoted above.

One explanation of the discrepancy could be that the Group II tumours in
Venice had increased parallel with the Group I tumours.

Such an explanation would mean that the agent causing the rise in lung cancer
in Venice would differ qualitatively from that operating in several other countries
examined (Great Britain, Finland, Norway), or tlie population would differ in its

response. , . ,

Another explanation could be a difference in the source of the materials studied,
and such a difference actually exists. In the Venice material 67 out of iS are
autopsj'^ cases, whereas in the Norwegian material there are only 36 out of 6 .
An examination of the male Norwegian autopsy cases reveals that here the ratio

is onl 3 '' 1-8:1. _ , ,

A second observation also indicates a fundamental difference in the
materials. Table II shows the age distribution of the Italian and the Norwegian

cases. _ . ., .1

In this table the Norwegian material does not include the carcinoias,
mucous gland tumours and the bronchiolo-alveolar cell carcinomas, because le

IA’NG cakckh in VlCNUn-,
Table U.—Age Dislribulion j« ^^<Ars

(■ill

Tumour

groups

Group 1
Group II

Total

Group I
Group II

Total

-SU

Age groups
(years)

Hn -r>'.'

Venire
4 U

1 3

Xorwny
55 1 35

0 27

213

-■511

211

135

155

Tol 111

511

403

740

tumours are nearly non-existent in the Italian material ami they often occur in
the lower age groups. Whereas two-thirds of the Italian cases are aged (.0 y an,
or more the figure is a little more than one-third for the >iorueginn. in spite of
the fact’ that the population at large shows a relatively higher representation of

old people in Norway than in Italy.

Finally, in Venice there is no thoracic surgical unit, and accordingly a c(‘rtniii
number of younger lung cancer patients go to Padova for .sjiecial (rcatiiieiit.
A certain selection of prognostically more favourable cases, probably including u
number of epidermoid carcinomas, may therefore have taken place, making the
Venice figures qualitatively less representative.

This preliminarj' study of histological types occurring in the Venice auto))Hy
material points to some fectors to be comsidered when comparative stmlie.s are
made.

The place of the lung carcinoma as the number one among the iiinlignant
tumours in males in an aggregate hospital with no special thoracic unit strongly
supports the conclusion that lung cancer in Venice truly is rather frcciueiit.

If this conclusion is correct, the Venice material has shown an uncxiicctedly
high number of Group II tumours, nearl}’’ e.xclusively consisting of adenocar-
cinomas. The absence of carcinoids, another peculiarity, may he incidental
because of the limited number of cases. Nevertheless, the low ratio Grouj) 1 -.Groii})
II tumours is remarkable and an e.xplanation mu.st he sought for. even if the ratio
;h2;l should most correctly he compared to the Norwegian autopsy fraction with
a ratio 1-8:1.

From a methodological point of view this small study underlines the necessit v.
when comparative studies of lung cancer types are made, to n.se materials of
similar character.

From the factual side the study indicates that a more extended future study
of the Vemce material is highly desirable, first to ascertain with greater cerh i^^t -
the true relative occurrence of the dilferent histological tjmes and nc4 f Im
pmsent findings are confirmed, to seek an explanation of the g St mmd or
adenocarcinomas as compared to the number of Group I tumouS

REFERENCES

Kheyb-erg. L.~(ia59) Acta Un. ini. Cancr., 15 , 78 .

612

THE HISTOLOGY OF Cn^INDROaiA OF aiUCOUS GLAND ORIGIN

A. C. THACKRAY and R. B. LUCAS

Froyn the, Bland-Svilon InsUtule of Pathologij, Middlesex Hospital, London, Kl, and Ik
Department of Pathology, Royal Dental Hospital of London School of Dental Surgery,

London, IV.0.2

Keceived for puMication October 27, 1900

It is noAv generally agreed that classification of mucous gland tumours on
histological grounds is a useful procedure since the various types of tumour
bear differing prognoses. Though there is still lack of unanimit 3 - ulth regard to
details of nomenclature, most pathologists and clinicians vould probabR' agree
to the recognition of the following neoplasms as distinct entities.

1. Mixed tumours. — These are the neoplasms traditionally described
as “ mixed salivar}" tumours ”. Typicallj^ thej' consist of an epithelial
component intimately associated -ndth mucoid or mj’^xochondroid inter-
cellular material.

2. Mucoepidermoid tumours. — The neoplasms described by Stewart,
Foote and Becker (1945), consisting of epidermoid and mucus-secreting
elements.

3. Cylindromas. — ^Tumours of characteristic architecture, to be de-
scribed in this paper.

4. Glandular, squamcncs and other carcinomas. — Frank carcinomas,
mostly glandular, sometimes squamous and occasionally of other t 3 pes,
comparable in behaviour to carcinomas elsewhere.

These four groups of neoplasms constitute the great majorit}'^ of aU tumours of
mucous glands and warrant separate recognition on grounds of structure and
behaviour. In addition, there are the less common tumours such as those of
the ox3q>luI, acinic cell and papillary C3''stadenom3 type, and the mesenchj’mal
tumours, wliich are not further considered here.

With regard to the behaviour of the commoner neoplasms, the mixed tumours
are generall}'' regarded as benign. How^ever, the 3 '^ show' a marked propensit 3 ' for
recmrence, due to factors such as the leaving beliind of peripheral nodules <h
growth after enucleation, the implantation of neoplastic cells in the tumour bed
and the presence of focal infiltrations (Pate 3 '’ and Thackra}', 1958). Apart from
recurrences due to such causes there is tire question of whether mixed tumours
can change in t 3 rpe to become carcinomatous. It is generally believed that sue i
changes do occur in a certain proportion of cases (Foote and Frazell, 195 ,
Frazell, 1954 ; Patey and Thaclcra 3 % 1958) but that on the whole they are in-
frequent. ,

The muco-epidermoid tumours, on the other hand, behave as maligna
grow'ths, both with regard to local invasion and metastatic deposits. The
dromas show a similar natural history, for they too are locally invaswe gror |s
and are also capable of giving rise to metastases. Their rate of growth, howeve ,

histology or cvlindkoma

I’, in

is generally

Se cylimlromn. .nul to iiulical<‘ tli.' w.,1,' vnn<-ly

of appearances ■which may be cnconutcicci.

2^omcnchiUirr.

In liis oriiiiimi e.'V‘’<'

Billroth in 1859 gave the first account of a cylimlroiim.
the neoplasm had invaded the orbit, probably from 1 lie mneons )

accessorj^ nasal sinuses, and he coined the name cylindroma beciuise the epitlu 1 m 1
elements of the gromth mere enclosed in wcll-dclined " cylindor.s of eoimectiw
tissue. Malassez (1883) mentioned some earlier casc.-^ that a]ijieare(l to tall inlo
the same group. The term “ basalioma ” was first used by Kroinpeelier (IhOS).
who considered the tumour to be of analogous nature to the basal cell growths
of the skin. Ahlbom (1935) and Ringertz (193S) also used the term basalionm.
In the more recent American literature the tumour has generally been luiown as
“ adenoid cystic carcinoma ”, a term apparently introduced by ICwiiig (I'oote and
Frazell, 1953), and series of tumours under this name have been rcjiorti'd by
Spies (1930) and others. Spies emphasized that the luinonr was a distinct
entity and should not be confused uith the adenoid cy.stic epithelioma of Ilrooks
and Tordyce, and that it was different from adenocarcinoma. Adenoeareinoina
of mixed tumour type has also been used as a designation (New and (3iildrey.
1931; Watson, 1935), and adenocarcinoma of cylindroma typo (I)ock(Tty and
Mayo, 1942, 1943; Quattelbaum, Dockerj' and Mayo. 194(5). Tlie original
term cjdindroma has come back into favour recently, and' l.onnox ( 1 9(5(() comments
“ I can find no better name than ‘ cj'-lindroma ’ for the group, (hough few members
contain any cylindrical structures ”.

Tumours of cylindromatous tj^je were at first regarded simply as variants of
the “ benign salivary gland tumour ”, as it was often called, anil were not, con-
sidered to differ in any significant manner from the majority of such tumours
Nevertheless, even early reports had shown that these neoplasms were \-crv ant
to recur after local removal, and could even metastasize (Ril)bert 19071' On
the other hand, some tumours which behaved in a malignant manner were thought.

of as raa,ig„.„s raix.d taraoora rathe, than 5re"o;iiS™te • raZIhS
til ; 't"^ inadequate appreciation of the structural distinctions between

cylinroml^clme trbrunSsTood’Tnr’af'^ structural characteristics of the
clinical behaviour. Montanus (1938) and MulHgS''7l''i3Trevim^^^^

licr

were

Distribution

“(i" “ “““gihg in the

ehe „,a, ea.t, idj

614

A. 0. THACKEAY ANV E. B. LUCAS

(1883) was one of the earliest authors to describe a tumour of the palate as
cyhndroma-like. A large number of reports of palatal mucous gland tumours
have since appeared though the neoplasms are very often reported as “ mixed ”
or “ benign sahvary ” tumours. The literature of these earlier accounts may be
found in Ringertz (1938). More recent reports of intra-oral salivary gland
tumours have given some prominence to the question of histological types and
most authors have stressed the necessity for distinguishing between “ mixed ”
tumours and the other varieties of mucous gland neoplasms. Recent series are
those of Rawson, Howard, Royster and Horn (1950), Russell (1955), Ranger,
Thaclcray and Lucas (1956) and Harrison (1956, 1957). The intra-oral glands
are the most frequent site of occurrence of cylindroma for though salivary gland
tumours in general occur less commonlj’- in the minor salivary glands the pro-
portion of cjdindromas occurring in those glands is about 15 per cent of all tumours
(Ranger, Thackra}'' and Lucas, 1956). whereas in the major salivary glands it is
in the region of 4 per cent (Foote and Frazell, 1953). In the case of the major
glands the parotid and sulrmaxillarj’^ are cliiefly affected, cylindromas of the
sublingual gland being verj’^ rare. Intra-orally, the glands of the palate and the
floor of the mouth are the main sites, about 70 per cent of all intra-oral cjdindromas
occurring in these two areas. The remaining tumours occur in the tongue,
the lip, or elsewhere in the buccal mucosa.

Cjdindroma also occurs in the respiratory tract vdth some frequency. In fact,
in a large series of tumours from all sites there are likely to be more neoplasms
from the respiratory tract, including the nose, naso-pharjmx and sinuses, than from
the salivary glands. Howe^^er, it is onl3'' comparatively recently that cylindroma
of the respiratorj’’ glands has been recognized as a distinct entity. Kramer and
>Som (1939) were the first to report a tumour of this type occurring in the bronchus,
and thej' give the earty literature for the tumour in other parts of the respirator)’
tract. Subsequently there have been numerous reports of such lesions, most
authors giving prominence to the question of differentiating the various t)pes of
neoplasm that ean arise from the respiratory glands. It is now generally agreed
that a clear distinction should be made between cylindroma, bronchial adenoma
of the carcinoid type and frank bronchial carcinoma (McDonald and Havens,
1948 ; Belsey and Valentine, 1951 ; Reid, 1952), though occasionally tumours
occur which appear to share characteristics both of the solid adenoma and the
cylindroma (Englebreth-Holm, 1944). In Enterline and Schoenberg’s (1954)
series, bronchial cylindromas proved ultimately fatal seven times as frequently
as “ carcinoid ” adenomas, and also recurred seven times as frequently.

CyMndroma also occurs in the laclrrymal gland, where it constitutes some 50
per cent of all tumours, the other 50 per cent being of the mixed type (God -
fredsen, 1948). Occasional breast tumours show the cylindromatous pattern
(Stewart, 1946), as do a number of skin tumours deriving from the sweat glands
(Lever, 1948).

Histology

General histological pattern.—The most commonly observed histological
in cylindroma is that shown in Fig. 1, the tumour consisting of feegu ar y s p
masses of cells in a rather scanty connective tissue stroma. .f

or alveolar spaces are present in the cell masses, giving rise to e cn
effect which is a very characteristic feature of this neoplasm. Houever,

HISTOLOGY or CYLIXDUOMA

t( 1 o

'M ahni'n! ,

it' i>r lln’

tumours show the cribriform pattern since the cystic spaces an* sfinieliinej
In such cases, the grou'th is of a solid type. Variations of tin* cystic or tin*
solid pattern account for much of tlic variation to he ohserved in tin* general
histological picture in c 3 -lindroma. though cliangcs in the stroma also eontrilmlc
in some cases.

The cystic pattern.— The cj'stic spaces in a cylindroma an* not all ftirmi'd in
the same manner. In Fig. 2. for example, the space re|)resents a duet, its w.all
consisting of a double row of cells arranged in a similar manner to those of a
normal salivarj* gland duct. The cells of the inner row, which will he refern'd
to as lumenal cells, are small, with oxvphilic cvtoplasm and a ve.-.icidar nneleii'
which contains a prominent nucleolus. These lumenal cells prohnhlv aho ha\-e
some secretorj* activity The cells of the outer row are larger, though’the nuele„.
IS smaller than those of the nmer row. The cytoplasm is marhedlv vacuolated
nd the nucleus stains darkly. XucleoH are rarely seen, 'riiese cells an* riinilar
n appearance to the myoepithelial cells of the normal duet . The material wi i
the lumen is granular and shows well-marked eosino,,hilia in scctir'nl ‘t - i,,;.d p .

p.sXZdTe'ifjjf" ri!' "[ ■«-

than the rule, for in the great maioritv of caL« is the e.vcciition rather

Thus in Fig. 3 the cjX

hy cells of the mjmepithelial tjme wliilst mostnni'^’^

by cells of the lumenal tjme. \ point of uoIa • "iilv

contents in these two type? of cyst 1 1^0 r ^ Y «l''-

contents are not markedly eosinophilic onr 'T' '"•'■"n’iHiclial cells the
Apart from arrangements of £ r l’-A..S.-t,ositive.

bounded by one or other of the cell nientioncd. where the evst.s are

m which there is a regular arrangePSnt'of ‘ '''Stances

s,'rs;v;s.r^-

the presence of a good deal of m'' ”T°epithelial ceH^rciio

As a further pUdJy them L'^vf niateriar Fi? 5

than proliferation of ceUs^ dilatation of the duct lil- '

like pattern shown in Fia proceeds to an evtremp f

to the formation of sW other cases tho ! ‘*'0 lace-

Wg. 5- Another ““ ""'I* Srf dnShe''"^”'”''

!>!■ •>uble ron- of Xdii“°1 '"‘“‘l’ <>f »ompre’ „d 1 % I

of "“y nlMresu’itf re**" '"led

manner
area at “A ”

616

A. C. THACKRAY AND R. B. LUCAS

becomes encircled in further sections, by epithelium, assuming an appearance
very similar to that seen at “ E Furthermore, the contents of cystic spaces
of this type are only very weakly P.A.S. -positive, in contradistinction to the snaces
lined b}" lumenal cells (Fig. 9). ^

These variations in histological pattern, rvith the differing modes of cyst
formation, account for the apparent i'screpancies in the staining reactions of the
mucoid material in the cystic spaces which have been noted by a number of
workers (Lemaitre, 1938 ; Ili-amer and Som, 1939,- MacDonald, Moersch and
Tinney, 1945 ; Belsey and ^^alentine, 1951). Eecentty, liowever, Azzopardi and
Smith (1959) have shoum that there are, in fact, histochemical differences between
the mucins in the different tj^pes of spaces. In the duct-like structures the mucin
is strongly P.A.S. -positive and is not metachromatic, whilst in what they term
the pseudo-acini (i.e., the spaces bordered by m 3 mepithelial cells) the mucin is
only u’eakl}" P.A.S. -positive but is stronglj^ gamnia-metaehromatic, showing
marked reduction of the metachroniasia after incubation with hjMuronidase.
The hjmJine material which appears to be formed in pro.ximity to the stroma is
P.A.S. -positive to a moderate degree and is rather less gamma-metachromatic.
Methjdene blue e.xtinction confirms these differences.

Comparable histochemical observations have been made in the case of mixed
tumours. Some authors, for example Hemplemami and Womack (1942), have
taken tlris to indicate that mixed tumours are of diploblastic origin, while others
(Grishman, 1952) consider the connective tissue type of mucin to be produced
by myoepithelial cells. The umrk of Erichsen (1955) and Cotclhn (1958) on
mammar}' neoplasms in the bitch prorddes additional evidence that mucin
produced bj' mjmepithelial cells is of what is generalty considered to be connective
tissue tjfpe.

Soil'd paUerns. — Solid tj^es of growth are much less common than the cribri-

form pattern and generally a part only of the grorrth is arranged in this manner,
the remainder being of the more usual arcliitecture. However, occasionally
an almost completely solid tj’pe of growth is encountered. Such growths may
be difficult to identify, though thorough examination will generally reveal small
areas here and there exhibiting more characteristic traits. Compression of
ductular structures, as in Fig. S, may lead to a practically solid type of growth,
but more often the appearances are due to the tumour cells forming conthmous
sheets. Areas of necrosis tend to occur in solid growths (Fig. 11). Mitotic

figures do not occur 'with anj^ frequency in either solid or cymtic tumours.

Two further uncommon variants may'' be mentioned. In one the cells are
predominantly fusiform or spindle-shaped, the appearance in some fields being
reminiscent of a neurinoma, with xvhat appears to be palisading of the nuclei
(Fig. 12 and 13). In the other the bulk of the tumour is composed of lumenal
cells in an acinar arrangement, Only^ in a few areas at the edge of the tumour
are myoepithelial cells to be seen grouped around the tubules and giving a clue
to the cyhndromatous nature of the growdh (Fig. 14).

Stromal changes. Stromal changes are sometimes prominent in cylindroma.
A rather scanty fibrous stroma is the commonest finding (Fig. 1) but often le
stroma is more abundant and frequently hyaline is present. The hyaline is
either changed stromal connective tissue or it represents a product of the umoii
cells, being laid down in proximity to the stroma. Hyalinization is often asso
ciated with a breaking-up of the cribriform pattern to form smaller ce group

histology OV CYT.IKDUOMA

The deposition of mucinous material in proximity
i ifcolf liv mucoid, is also not unco

the mucoid material, as occurs so frequenth , and tj pIcall^

Thus in Fig. 16 the cell groups have quite distinct outlines, aiu
iims 111 i S „.u;„i, Jo nlninst ciitirclv imicoid.

to the stroma, or

However,
merge with
mixed tuniouis.
and are ele.arly de-
Oceasioimlly.

Thus in Fig. 16 tlie ceii groups lum. , . ,

marcated from the stroma, tvhich is J

hon'ever there are encountered tumours in which the cpitheMl ‘ “f 1 « ’

‘Cl^ ”’into the mucoid in much the same way as occurs ,ii mixed tun o r.
This is shorvm in Fig. 17. and much of the tiniiour from winch tins section u.is

prepared showed similar changes.

eVst formation in the stroma has already been discu.sscd.

Differentiation between cylindroma and mixed htmour . — In the great majority
of cases there is no difficulty in differentiating between tyjiical cxamjiles of cylin-
droma and tjqiical mixed tumours. Occasionally, however, instances occur
where the distinction is not so readily made. One source of jiossililc' confusion
is the mucoid change which is a tjqiical feature of the mixed tumour and .some-
times also occurs in the cylindroma. However, the distingiii.shing feature, as
already pointed out, is the clear demarcation of epithelial cells from mucoid intiw-
cellular material in cjdindroma, whereas in mixed tumour the cjiithelial cells
appear to blend with the mucin. The occasional cylindroma in which imicoid
intercellular change appears also to involve the epithelial cells, as sliown in I'ig. 17.
maj^ be confused with mixed tumour. A very umisiial exanqilc of Ibis type of
mucoid change in a cylindroma is shown in Fig. IS, The tumour, from the paro-
tid, showed on macroscopic examination a small nodule of different ajijicarance
to the rest of the growth, measuring just under 1 cm. in diameter and situated at
one pole. On section the greater part of the tumour showed the tyjiical cribrifonn
pattern of cylindroma but the structure of the nodule, on preliminary examinat ion,
appeared very similar to that of a mixed tumour, consisting of scattered groiijis
of epithehal cells and ductular structures in a completel}’ mucoid matri.x. Soria!
sections of the entire area showed the nodule to be clearly deinarc<atcd by a (list inct
fibrous capsule from the rest of the tumour, with which no connection could be
demonstrated. However, more detailed examination showed the cells to bo of
material clearly demarcated from the surrounding mucoid

Another tj^e of configuration in cylindroma that shows some similaritv to
that seen m mixed tumour is the pattern illustrated in Fig. 5 . Tliis is comnarable

cystic ”, '»d

anioiiiit. Duct formation in cylintaa (pj 7 ° marata mor'”"’'

very similar to that seen in some miverl ^ Produce appearances

ery similar to that seen in some mixed tumours.'^

given

same

618

A. C. THACKRAY AND R. B. LUCAS

type of course. This tends to be rather prolonged and is characterized by slow
but persistent infiltration, growth along nerves often being a prominent future
Metastases occur relatively late in the course of the disease and the secondary
deposits generally show the same histological appearance as the primary growth.
The same applies to local recurrences, even after a number of years. In some
cases, however, local recurrences or metastases may show quite a different picture,
presenting as spheroidal cell carcinoma from the histological point of view, and
clinically assuming a more frankly carcinomatous course. Fig. 19 is an example
of this tjqje of groudih. The primar}' growth, in the trachea, showed the typical
cribriform pattern. The section illustration was from a local recurrence.

Histogenesis

Theories of origin of mucous gland tumours in general have been dealt with
extensively in the literature, but there is very little ■with regard to cylindroma in
particular.

An early view "was that of Krompecher (1908), who considered the tumour
to be akin to the basal cell carcinoma of the skin, both clinically and patho-
logicallj'-, and for this reason he used a corresponding nomenclature. Other
authors have also referred to cylindroma as basal cell carcinoma or basalioma,
either because such terms have appeared descriptively appropriate or because
they have attributed a mucosal basal cell origin to the gro'vdh (Beck and Gutt-
man, 1936). However, most authors who deal -with the question of origin are
agreed that the cjdindroma arises directly from mucous, salivary or other glands,
though there is some doubt as to which elements of the glands are responsible.
This view is supported bj'' the finding of tumour originating, or apparently ori-
ginating, from gland elements, though reports of this are remarkably scanty,
considering the number of tumours recorded in the literature. However,
McDonald, Moersch and Tinney^ (1945) state that in one of their six cases the
tumour appeared to be arising from mucous glands in the broncliial wall and
Reid (1952) has also seen tumour originating in the duct of a broncliial gland.
Russell (1955) suggests that the tumour arises or appears to arise from dilated
mucous gland ducts.

In the neoplasms reported in this study, the close proximity of tumour to
relatively normal mucous glands was noted in a number of cases. Mostly there
was a clear demarcation between tumour and normal tissue, but sometimes grov i
and gland were intermingled. In such cases it might well have been the case
that normal glandular tissue ivas being invaded by tumour, rather than gnmg
rise to it, though in one lesion there appeared to be an actual transition be veen
tumour tissue and normal gland.

The so-called “ transitional lobules ” have been thought to constitute
tional stages between normal glandular tissue and tumour. These lobu ^ a
aggregations of glandular tissue showing some dilatation of the ducts mr
generative changes in their lining cells a'nd in. the cells of adjacent alveoli.
inflammatory infiltration is present in the stroma. These changes . j

described in connection with mixed tumours though not so far as can be asce ^
in cylindroma. Fig. 20 shows an example of tins tyqie of change m one
cases, hut we agree 'with Ringertz (1938) that the changes are degenera n
not neoplastic.

histology OV C.Vl.lKOUOMA

r.i;i

The expenses
Cancer Campaign

of this invostigntion have been clofrayo,! l.y (ho Hrilisli Ki"l'!n'

REFERENCES

-(ItlKi)

AmnoM H. E (1935) .4d« -Rarfio/., Suppl. 23.

Azzopae’dTj (4. AxmLmi.O.D.Mlh59).C 13K

Beck, J. C. axd Guthiak, M. R.-{193G) Am OIoL cIc hi.

Belsey, R. H. R. axd VaVLEXTiXE, J. G.— (Iflal) -T ■ I f'th. BnrI.. 63. .1/ /.

BiLEROTH, T. — (1859) Virchows Arch., 17, 357.

COTCHIX, E.— (1958) J. comp. Pol/i.. 68, 1. n, , , r-s moo

Dockerty, M. B. and Mayo, C. 1V.-(1W2) ««’'£!•

Surgery, 13, 416.

Engeebreth-Holm, j. — (1944) Ada chir. scand., 90. 383.

Ekteruxe, H. T. axd Schoexberg, H. W.— (19.54) Cancer, 7, 003.

Erichsen, S. — (1955) Ada. path. microbioL scand., 36, 490.

Foote, F. \V., Jr. and Frazell, E. L.— (1953) Cancer, 6, 100.5.

Frazeel, E. L.— (1954) Ibid., 7, 637.

Godteredsen, E. — (1948) Brit. J. Ophthal., 32, 171.

Grishman, E. — (1952) Cancer, 5, 700.

Harrison, K. — (1956) Ann. E. CoU. Surg. Engl., 18, 99. — (19.57) Ann. Otol,. etc., El.
Louis, 66, 459.

Hexiplemann, L. H., Jr. and IVoxiack, N. A. — (1942) Ann. Surg., 116, 3-1.

Keaxier, R. and Sou, M. L, — (1939) Arch. Otolaryng., Chicago, 29, 356,

Rboxipecher, E. — (1908) Beitr. path. Anal., 44, 88.

Lexiaitre, Y. — (1938) Ann. Oto-laryng., 57, 185.

Lennox, B. — (1960) in Recent Advance.s in Pathology ’, 7th Ed., edited In’ G.

Harrison. London (J. & A. Churchill), p. 9.

Lever, W. F.— (1948) Arch. Derm. Syph., AM'., 57, 332.

McDonald, J. R. and Havens, F. Z.— (1948) Surg. Clin. A'. Anicr., 28, 1087.

Idem, Moersch, H. J. and Tinney, W. S.— (1945) J. Ihorac. Surg., 14, 445.

Malassez, M. L. — (1883) Arch. Phys. norm, path., 3me .serie. 1, ISO
Montands, W. P.— (1938) Surgery, 4, 423.

Mulligan, R. M.— (1943) Arch. Path. (Lab. Med.), 35, 357
Nexv, G. B. and Childrey, J. H.-(1931) Arch. Otolaryng., Chicago, 1

Patey, D.H. AND Thackray, A. G.— (1958) HriL. 7., W 45 477

Quattleb^i, F. W., Dockerty, M. B. and Mayo, C. W.-(mG)'sura. Cynec. Obdd,.

- Vi i'ou; jjric. .y. uaiicer. lU, ].

', J. M., Royster, H. P. and Horn, R. G., Jr.-(] 950) Cancer,

Nexv, G.^B. and Cidddrey, J. H.-(1931) Arch. dloiaryng'., Chicago, 14 609

n /TA=rov n .’t J o -- ' ’ *

•/. hurg.,

L C. IV.-

Raxger, D.,^ackeay, A. C. AND Lucas, R. B.— (1950) Brit ./ Cnnrer in i

BaA\SOX, A. J., HO'WAED, J. 51., Roystt-.k TT P A-v-r. t> r/ I. ' ' *

3, 445.

Reid, J. D.-{1952) Ibid.. 5, 685.

Ribbert, M W. H.-(1907) Dlsch. med. Wschr. 33 126

f 'f sSpi 2- ■

kdssell, H.— (19o5) Bnt. J. Surg., 43, 248 “

. 2.. soa.

620

A. C. THACKRAY AND R. B. LUCAS

EXPLANATION OF PLATES

Pig. 1. — A commonly occurring pattern in cylindroma. The tumour consist.? of cell masses
containing cystic spaces, giving rise to n cribriform appearance. xGO.

Fig. 2. — High yiowcr view of n cylindroma, showing the two cell types arranged as in a nonnal
mucous gland duct. x330.

Fig. 3. — To show the two types of cellular arrangement around cystic spaces. x250.

Fig. 4. — duct lined by luinonal cells at the centre of the field, with myoepithelial cells above
and below, x 330.

Fig. 5. — ^The cells in this field are nearly all of myoepithelial tjTie, with a considerable amount
of intercellular mucoid material, x 05.

Fig. G. — A tj'pical appearance in cylindroma ; well-marked cribriform pattern. x45.

Fig, 7. — ^To show the formation of rather irregular duct-like spaces. xOo.

Fig. 8. — To show compressed duct-like structures, x 05.

Fig. 0. — Darkly stained contents of lumenal cysts contrasted with weakly P.A.S. — positive
stromal cysts, x 185.

Fig. 10. — Mode of formation of stromal cysts. “A ” indicates an area of stroma about to
become enclaved bv epithelium ; “ B ” indicates a similar area, eompletel}' encircled.
Xl85.

Fig. II. — Area of necrosis in a predominantly solid growth. xGO.

Fig. 12. — Bundles of elongated cells reminiscent of a neurinoma in the upper part of the field,
with more tj'pical cj'lindromatous pattern below, x 100.

Pig. 13. — ^High power view of the spindle cell area in Fig. 12. x 190.

Fig. 14. — Field at the edge of a tumour which for the most part was composed of cells of
lumenal type, x 190.

Fig. 15. — Breaking-up of the cribriform pattern associated with hyalinisation. x45.

Fig. 1G. — Sharply defined cell groups with inteiwening mucoid stroma. xDo.

Fig. 17. — Epithelial cells in a cylindroma appearing to melt into the mucoid. X95.

Fig. is. — D istinct nodule of growth in a parotid cylindroma. x3.

Fig. 19.— Anaplasia in the local recurrence of a formerlj’ well differentiated cj’hndrom.'i.
X95.

Fig. 20. — A “ transitional ” lobule of mucous glands, adjacent to a cj’Imdroma. X.lo.

British .Toi'rn'ai. or Cantkr.

Vol. XIV. Xo. .1.

T'larliniy mid

Vnl. XIV. N... I

Biirnsii .loriiNAi. or Can(;i;ii.

LY5IPH0SAEC0JU IN THE OVAEY IN VOUKO AI'EICAN HIHLS

D. StJ. brew axd .1. O. JAGKSOX
From the Devarlment of Pathology, University College. Iha-lan. ^ igerin

Eeceivcd for publication September 30, lOliO.

Lyjiphosaecoma is generally considered to he a disease prcflo.ninantiy of
middle and old age though it is not rare in youth. The occurrence of 1} in])!iosa
coma in young Mrican girls involving the ovaries as the most conspicuous site i.s
of such freque^y at University College Hospital. Ibadan, as to warrant description.
During the years 1958 and 1959 the total number of malignant tuinoiii-s among
1286 post-mortem examinations at this hospital was 1 07. The_diagnosis of lympho-
sarcoma or reticulum cell sarcoma was made in 25 cases and in 7 of those the jiatieiits
were girls under 14 years old.

Table l.—Age and Sex of 25 Post-mortem Cases of Lymphosarcoma and
Reticulum Cell Sarcoma

■’Adult"
•Age not

1-4 5-14 15-24 2.5-34 35-44 4r>-r.4 stuted

0 .5 .1 .2 .5 .0 .1

0 .7 .0 .2 .1 .1 .0

Age in
years
Alales .
Females

Lymphosarcoma was the commonest of the 17 malignant tumours tliat were
found at necropsj' in children. Other tumours seen ivere 1 sarcoma of the urinary
bladder, 1 medulloblastoma, 1 retinoblastoma, 1 \Shlms tumour and 1 astroc}'toina.
In only one of the seven girls under 14 years with lymphosarcoma were the ovaries
unaffected. No tumours of the testis were found in boj^s. The main clinical features
and the necropsy findings in the six African girls ivho had ovarian tumours which
proved to be lymphosarcomas were as follows : —

Case 1

S.S. (21640), aged 5 years, was admitted to ho.spitai because of proptosis of the
right eye 5nth ophthalmoplegia and bilateral masses 5 vhich were palpable in the

TbT' TJ" the posterior clinoid pro

ce.sses. The haemoglolnn concentration was 8-2 g. per 100 ml and the leiionputo

“I"*

-urfacc of i brain le" SteVr S'

m the pituitar}' fossa extending into the orbit on the rEt / tumour tissue
»■«. reapeciinei. Tbe

622

D. STJ. BREW AND J. G. JACKSON

to, but separate from the right ovarian mass there was a large diffuse retroperi-
toneal sarcoma extending from the pelvis towards the liver. The lymph glands of
the thorax and abdomen were not enlarged. The stomach and intestines were
normal. The spleen was enlarged but was of normal appearance. The liver, hidne 3 's,
ureters, bladder, pancreas and adrenal glands were normal The heart and luiif^s
were normal. ”

Case 2

M.A. (20059), aged 8 years, had been paraplegic for 8 days. X-raj^s showed a
shadow at the level of the fifth thoracic vertebra suggestive of a para-vertebral
abscess. The haemoglobin was 8 g. per 100 ml. and the white cell count 5400 per
cu.mm, with a normal differential count. A costo-transversectomy was performed
atrd at operation enlarged lymph glands were noted. The patient deteriorated
steadil 3 ^ Seven weeks after operation a pathological fracture of the right femur
occurred and there was also a seeondarj' tumour in the right tibia. She died the
next day.

Necropsy . — There was diffuse infiltration of the bone and soft tissues of the
upper thoracic spine by soft white neoplastic tissue. Mediastinal tymph glands
were enlarged. The heart and lungs showed no abnormalit 3 ^ Both ovaries were
completel 3 ’' replaced b}' soft ■white tumour tissue ; each measured 7 cm. in diameter.
The rest of the genital organs "were normal. Both kidnej's were greatl 3 " enlarged
and were almost entirel}'- replaced b}' soft white tumour tissue (Fig. 1). The adrenal
glands were replaced b 3 ' tumour. All the para-aortic lymph glands were greatly
enlarged. The liver and spleen were normal. The surface of the brain showed a
slight flattening of the convolutions. The pituitar}’- gland -n^as replaced b 3 '^ a large
deposit of tumour wdiich extended into both cavernous sinuses.

Case 3

M, J. (22643), 6 years old, wms admitted because of a swelling of the left upper
jaw. The submandibular and cervical glands were swollen and the spleen rvas
enlarged. X-rays shorved a tumour in the left maxilla and an osteoljTic soft tissue
tumour in the central area of the mandible near the syraph 3 ’^sis menti. Haemoglobin
10-7 g. per cent, leucocyte count 8000 per cu.mm., pol 3 miorphonuclears 69 percent,
l 3 "mphocytes 27 per cent, eosinophils 4 per cent.

Necropsy . — There was a nodule of white tumour 1-5 cm. in diameter in the
anterior ■n'-all of the left ventricle. The lungs were normal. The mediastinal glands
■were not enlarged. The left kidney -was replaced b}’^ a large homogeneous mass o
tumour ; the right kidney contained several tumour nodules up to 2 cm. in diameter.
The ovaries w^ere 7 cm. and 8 cm. in diameter and consisted of homogeneous win e
tissue (Fig. 2, 5). The spleen contained several -well defined tumour noduies.
Lymph glands were enlarged in the splenic hilum and in the transverse mesoco on
but not elsewhere. The skull and brain rvere normal.

Case 4

A. A. (25487) aged 8 years, had had oedema of the left leg for eight weeks an^
was admitted to hospital noth a discharging sinus in the groin at the si e o «
biops 3 \ There was a mass in the right iliac fossa and a nodule was presen on
bridge of the nose. X-rays showed diffuse involvement of the diaphysis o e

LY^ilPHOSARCOMA IN THE 0\ AK^

tibia and fibula. Haemoglobin 11-5 g. per cent, leueocyles 5:100 per cu.innn

were normal.

Eoti o Ss n-efe enlarged to about S cm. in diameter with .smooth g ■«tomng
surface and were composed of homogeneous white tumour ti.ssue (I'lg. • ). o i
kidneys contained many discrete white nodules n]> to 2 cm. m (hameter (1 ig. • ).
The p\ra-aortic Ijunpirglands were slightly enlarged and of uniform white con-
sistency and there were seyeral enlarged glands in the left mgumal region. J lie
mediastinal glands were normal. The liyer was slightly fatty : the spleen uns
normal. The nodule in the bridge of the nose consisted of a white tumour lying in
the subcutaneous tissue and eroding the underlying bone.

Case 5

F. A. (36731). 9 years old, gaye a histoiy’ of abdominal swelling for two years
and had deyeloped pain in the abdomen recently. There were niiilti-lobiilatcd
masses arising from the pelyis and reaching the umbilicus. X-rays shoued no
tumours in the bones nor in the chest. Haemoglobin 7‘8 g. The lciicoc\tc count
was not done. Xo abnormality was noted on a thin blood film. A laparotomy was
performed and inoperable ovarian tumours were found.

Necropsy . — ^The heart and mediastinal lymph glands wore normal. There was
basal bronchopneumonia in the right lung. Two large solid masses in the pelvis
replaced the ovaries. The right was 25 cm. and the left 10 cm. in diameter. The
lymph glands alongside the abdominal aorta were enlarged. The kidneys were
normal and no abnormalitj’’ was present in the liver, .spleen, adrenals or jiancreas.
The skull, brain and meninges were normal.

Case 6

A. Y. (39824), aged 11 years, was an ill, pale, wasted child who complained of
a swelling on the left side of the abdomen which was said to liave been first noticed
25 da^'S before admission. On examination there was a large mass in tlie left upper
abdomen and several other smaller masses elsewhere in the abdomen. X-rays of the
chest and of the jaws were normal. An intravenous pyelogram showed distortion
of the renal pelvis on both sides. Haemoglobin 7-4 g. per cent ; leucoeyte count
11,500 per cu.mm, with polymorphonuclear leucocjhosis.

Xecropsy.— The thjwoid gland was difiusely infiltrated by white tumour tissue :
the heart was normal and the lungs showed congestion. The mediastinal glands
Mere not enlarged The mass m the left hj^jochondrium consisted of the enlarged
congested spleen together with a large diffhse mass of white tumour tissue hdui
m the omentum between the spleen and the stomach. There w^ere several infarcts
m the spleen. The para-aortic and mesenteric glands were greatly en arTed TWl!
kidneys were enlarged and the greater nart of their J ^ enlarged. Jloth
numerous nodules ofwhitetumoS The substance was replaced by

masses (Fig. 4). There was also a deon-sit

livpogastriLi. Thel^er isS^^^^^^ T tlie

tSionloth”’ oT" ™ ntmT

two and a half years thelxds°ed™ariS"ofoySn ^

G24

V. STJ. BREW AND J. B. JACKSOK

Taele II. — SHe of Tumour Deposits in Six Post-mortem Cases

Case number

3 i

Cranium

-f

.Taws

Other Bones
Lymph Glands

—

Abdominal .

"T"

-L

Peripheral .

—

Kidneys .

A-

Adrenals

Spleen

—

Liver

—

Heart

Thyroid

—

Ovaries .

-f

-1.

ovarian tumours. In six cases there were tumour deposits in sites other than the
ovarj^ ; one had paraplegia due to an extra-dural deposit in the spinal canal ;
in another case the intra venous pyelogram showed alteration of the pattern of the
catyces suggesting deposits in the kid^nejn The blood count n^as done in all but
one of the cases ; there was no evidence of leulvaemia. Tliree of the patients have
not been seen again since thej" left hospital. One died two days after leaving
hospital, one was moribund when seen six weeks after operation and two had
multiple tumours when last seen. One patient had an ovarian tumour 20 cm. in
diameter histologicall}'^ identical to the other cases. A right ovariotom}"^ was done
and ten months later she was well with no er'idence of tumour on clinical examina-
tion. Only two other ovarian tumours from 3 ’’oung girls were examined histologically
in the same period. Both were cj'stic teratomas.

Histologicallj' the tumours were similar in ever}' case. The}' were composed
of sheets and masses of cells with small round h}'perchromatic nuclei and scanty
cystoplasm. The tumour cells lay in a reticulin network. Patches of necrosis
u'ere seen in some places. There rvas some slight variation in the size, shape and
depth of staining of the nuclei and moderate numbers of mitoses were present.
Scattered tlnoughout the tissue there were larger reticulum cells with single
nuclei and abundant pale-staining cytoplasm. In places because of shrinkage of
the reticulum cell, or absence of its nucleus due to artefact, a false appearance of
rosettes was formed. No neurofibrils could be detected. Normal ovarian tissue
appeared to have been completely replaced, no remnant being seen in any of the
sections. The tumours in the lymph glands, kidneys, and other sites presented
an identical histological picture. Tlie histological appearances were those of
lymphosarcoma.

EXPLANATION OF PLATES

Fig. L — The enlarged ovaries, the kidneys and the femur of case 2. i natural size.
Fi<j. 2 . — Section of the ovar^' of case 3, H- and E- X 145.

Fig. 3. — Section of the ovary of case 4. H. and E.

Fig. 4. — Section of the ovar^^ of case G. H. and E. Xl45.

Fig, 5. — Ovaries of case 3. Natural size.

FxG. 6. — ^Kidneys of case 4. Natural size.

British .Toi-rnai, ok Canck.ii.

V.il, XIV. N*.,. I

son.

nnd Jack,

British JornsAi. or CANcr.it.

Vol. XIV. No. I.

Brftw and ‘Tnokson,

lymphosarcoma IX THH 0\ AlH

DISCUSSION

Tt well recognised that Ivmphosarcoma is coiniiinii in tropical Africa

It IS .‘u„f 5.3 per cent of 10(1(1 tumours in ^Igcrla

Elmes and Bal nin ( ’ 'j . p^j-^edtliat Ivinpliosarcoina and rcticu-

were lymphosarcomas I

lum cell Barcomatogether formed 01 rauminnOo-n found !.•(■, per cent.

recorded by the Kampala Cancer Kegistry, tumours 01 me 'ym -m-*- •l'."
about twice as common as would be expected by comparison w. ’

of the combined lymphatic malignant diseases was less m .Ncgroms vuau m . ai ee-
soids The frequency with which lymphosarcoma occurs in young African clnldrcn
has been noted by O’Conor and Davies (1060) who state that during the seven
years 1952-1958 the Kampala Cancer Registry recorded 125 malignant tiinuuirs
occurring in children up to the age of U years among wliich tlicrc were 57 cases
of Ijunphosarcoma. Further, Rosenberg, Diamond, Dargeon and Graver (I95S)
point out that when lymphosarcoma occurs in childhood, extra-nodal sites " appear
to be primary ” more frequentty than in adults, although in tlicir scries of cases
there were no negroes.

We are not aware of any report dealing with the frequent occurrence of lynijiho-
sarcoma in the ovary in childhood. The condition appears to be uncommon in
Europe not being mentioned by Harnett (1952), Haines ( 1 958) or Symmers ( 1 958).
In a re\dew of the literature pertaining to ovarian hmiphosarcoma Kelson.
Dockerty, Pratt and ReMine (1958) found only 4 cases of “ so called primarv ”
and 24 cases of obviously secondary disease. They added G cases of their own* in
which they considered that the chnical features pointed to an ovarian jiarticijiat ion
in a more generalised lymphoblastomatous process. Kone of the cases described
occurred in children. Burkitt (1958) reported from Kampala 38 cases of a tumour
involving the jaws of African children which O’Conor and Davies (1960) now
consider to be lymphosarcoma. There were deposits in the tlnnoid, testis heart
stomach, salivary gland, cranium, femur, liver and kidneys, but ovarian tumour.s
are not mentioned in any of the cases. Davies (1959) mentions 5 cases of ovarian
tumour occurring among 179 childhood cancers but the histological diagnosis of the
ovarian tumours is not given. Two undifferentiated round cell tumours of uncertain
natme which occurred in the ovaries of girls aged 10 and 15 years were reported

wh?r'\^ nTr Edington (1956) mentions two girls aaed G^r'ears

who had bilateral ovanan tumours which were considered to be dysaerniinouir

0 Conor and Davies (1960) do not mention the ovaries in an anah^i^ nf ?“• ’

dence of involvement of different organs in 18 anlnTtcior? r ? * ^ Jiici-

in chhdhood. In a report of a Cancer Sunmy in the Tran^vaTwi

and Oettle (1960) did not find evidence of a high Lque^crof

has been reported from East Africa They found of lymphomas such as

giristhathavebX^i"^^^^^^^^

626

D. STJ. BREW AND J. G. JACKSON

sarcomas ; among the seven girls who died in hospital from lymphosarcoma there
was only one case in which the ovaries were not affected. During the same period
necropsies were done on 4 adult women who had died of lymphosarcoma and 107
ovarian tumours from adult women were sectioned. Among these no case of
lymphosarcoma involving the ovary was found.

In the absence of a census and civil registration of death we are not able to
make any exact estimate of the frequency of ovarian lymphosarcoma in the
population but we believe that tlie frequency of the disease in the post-mortem
and biopsj’^ material of this hospital suggests that it is more common in jmung
African girls in Nigei-ia than has been recognised hitherto and that this may well
be true of other parts of tropical Africa.

srorMARY

Involvement of the ovaries is a common and conspicuous feature when lympho-
sarcoma occurs in young African girls in Ibadan, Algeria. Tlie majority of ovarian
tumours in young girls here are l 3 '-mphosarcomas. The same is not true of adult
women.

We are grateful to the medical staff of the Department of Obstetrics and
Gynaecologj' and to other surgeons and phj^sicians for permission to use their
clinical records, to Dr. W. P. Cockshott for radiological reports and to Sir. P. E.
Speed of the Medical Illustration Unit, University College Hospital, Ibadan, for
the photographs.

REFERENCES

Bubkitt, D. — (1958) Bril. J. Svrg., 197, 218.

Casiaxn, R. — (1954) Bull. Soc. Pat. exol., 47, 614.

Dawes, J. N. P. — (1959) in ‘ Modern Trends in Patholog.v ed. D. H. Collins, London
(Butterworth & Co.), p. 153.

Idem AND Wilson, Barbara A. — (1954) E. Afr. med. J., 31, 395.

Idem, Wilson, B. A. and ICnowelden, J. — (1958) Brit. med. J., 2, 439.

Edington, G. M. — (1956) Brit. J. Cancer, 10, 595.

Elmes, B. G. T., and Baldutn, R. B. T. — (1947) Ann. trap. Med. Parasit., 41, JM-
Haines, M.— (1958) in ‘ Cancer ’, ed. R. W. Raven. London (Butterworth & Co.), 'oi.
9 p 298

Harnett; W. L.— (1952) ‘ A Survey of Cancer in London ’. London (British Empire

Cancer Campaign).

Higginson, j. and Oettle, A. G. — (1960) J. nal. Cancer. Inst., 24, 589.

Nelson, G. A., Dockerty, M. B., Pratt, J. H. and ReMine, W. PL .—( mb } Amer . .

Obstet., Gynec.

O’Conor, G. T. AND Davies, J. N. P.— (1960) J. PediaL, 56, 526.

Rosenberg, S. A., Diamond, H. D., Dakgeon, H. W. and Cbaveb,

New Engl. J. Med., 295, 505.

Steiner, P. E.—(1954) ‘Cancer: Race and Geography’.
Wilkins Co.).

Symmees, W. St C. — (1958) in ‘ Cancer ed. R. W. Raven.

Baltimore (Williams &
London (Butterworth &

Co.), Vol. 2, p. 448.

W. P.

From the Royal Marsden HoxpUal, lyondnn. .S.U .3

Received for puWiont ion ScptomlwT 11. UIOO

Ix patients Arith advanced cancer, it is often not only (bnicu t to prolonj, liR .
tut als^o to be certain whether treatment has done this. 1 he relief of sy mpl «mis.
on the other hand, is easier to assess and in many cases not so difiicult to nc.com-

^^"^Huegins and Scott (1945) described the effects of adrcnalccitomy in breast,
cancer This operation apparently prolonged life and often relicvecl snffcnng.
Soon, however, it became evident that this only occurred in a comparatively small
number of patients. The proportion of remissions obtained in the nccouiit.s of
published cases was never more than 40 per cent. The fact that so many pat ieiit s
were unaffected by adrenalectomy was explained, houeccr, b\ the suggestions
that the tumour was either not “ hormone dependent ”, or had become auto-
nomous. Alternatively, there may have been incomplete removal of the sources
of oestrogen production which may be either in accessory adrenal or ovarian
tissue. In all the patients who benefited from the operation, reactivation of the
disease occurred, in the majority of cases, within two years, though, excejitionally.
a few remained well for periods as long as three or four years. The simplest,
explanations for this reactivation were that there had been a continuation of
oestrogen secretion, or that the tumour was now being stimulated by pituitary
hormones, or possibly, that it had become completelj^ autonomous.

lYhen, therefore, Luft and Olivecrona (1952) described the results of treatment
of cancer of the breast by hypophyseetomy, it seemed possible that this treatment
might give better results than adrenalectomy. The more complete removal of
oestrogenic hormones, as well as prolactin and somatotrophin, should, theoretic-
ally, raise the remission rate very considerably. It was necessarj^ to accept the
fact that adrenalectomy and hjTiophj-sectomy should only be undertaken in
advanced cases for palliation and not used in the early cases as a curative pro-
cedure. The aim of treatment, in the advanced case, was to provide amelioration
of the disease, with rehef of symptoms, for trvo or even three years. It was
decided, therefore, that only advanced cases, in whom all recognized palliative

lijTiopliyscctom/is'Scult ™<1 eomplclc

<l.e pitaita,y 4 interatS “> <lioy

628

W. P. GBEEKDfG ET AL.

History

Tlie historj' of irradiation of the pituitar3'^ goes back tliirtj- j-ears or so
Henderson (1938) in reporting on 338 cases of pituitarj- adenomata treated bv
Cushing, described the implantation of radium, bj^ the transphenoidal route, into
patients witli basophil adenoma. In 1936, Lodge described an approach Ha tlie
orbit and ethmoid sinus to tlie sella turcica, in cases where the latter had become
grosslj" expanded by a tumour ; after removal of as much of the growth as possible
radon seeds were implanted (Northfield, 1949). Tliis same route is used by Bauer
(1956) to insert a cannula into the gland.

External irradiation with com'entional X-rays produces little or no effect
upon this extremely’- radioresistant structure (Kelty et al, 1951), a high energy
proton beam can destroys the gland (Tobias el ah, 1958).

Other methods such as electrocoagulation and the injection of chemical or
radioactive colloidal solutions, have also been tried. Electrocoagulation (Bauer,
1956) has not proved satisfactory^ because the electrode becomes covered with an
insulating lay^er of charred tissue and further destruction is thus prevented.
The injection into the gland of solutions of corrosive material, or of radioactive
materials is dangerous because of the impossibihty of limiting the fluid to the
confines of the sella.

Badon seeds were the obvious choice, but experience has shown that their
penetrating gamma ray^s cause serious damage to the optic nerve which may-
lead to total blindness in some cases (Forrest et al., 1956 : Westminster Hospital
Beport, 1956). It seemed reasonable, therefore, to try the effect of radioactive
gold grains (gold- 198) and these were first inserted in February 1955. Tliis
comparatively simple procedure from a technical point of view should be suitable
for use in a seriously ill patient. Since the expectation of life following implanta-
tion would be unlikely to exceed three y-ears, it would appear justifiable to accept
the possible occurrence of delay^ed effects of irradiation following damage to the
hypothalamus.

Basmussen, Harper and Kennedy^ (1953) suggested from experimental evidence
that y'^ttrium 90, a pure beta ray emitter, would be a suitable source for producing
localized destruction of the pituitary^ udthout damage to the surrounding structure.
Their experimental findings were confirmed by' Yuhl et al. (1955) who introduced
y'ttrium pellets at craniotomy'. Yttrium was therefore used for this purpose at
the Boyml Marsden Hospital in July' 1956.

BetAveen February' 1955 and March 1958, 100 patients rvith metastatic carci-
noma of the breast were treated by' implantation of the pituitary. Gold-19S
was used in 54 cases and y'ttrium-90 in 36. The remaining 10 patients had first
one, and later, the other isotope implanted. Screened gold grains 2-5 mm. long,
0-8 mm. diameter and with a sheath of platinum 0-15 mm. thick were used at
first, but later, unscreened rods 5-0 mm. long, and 0-8 mm. diameter Avere im-
planted. . ,

Gold-198 emits mainly gamma rays which are less penetrating than those
from radon and a small quantity' of beta ray's AA-hich do not penetrate, for le

EXPLANATIOJf OE PLATE

Fig. 3. — The pituitary occupies about three quarters of the depth of the fossa in this case.
Fig. 4. — Scaphoid anterior pituitary lying on the floor of the fossa.

pituit.^by ibrapiatiox vou nURASl

• (Viii n

””'

vnt]\ that from gold.

. , . „„^icr haclci lost lit ic niid ”"’'’’‘'’l'r

jr srr%"f f t;S;trc“L:ri:;r r

"jMIl'ciA — .«t«l with .trc,.l.>n.yo... ■ -r ■

Fig. 1.— Isodose curves for "Y and ”’Au. (Doses equated at 3 min. from source.)

for two or tliree days before operation, but this was discontinued later in (lie
series. Cortisone 50 mg. daily was started on the daj' of operation ; in the earlier
cases it was witheld until signs of adrenal insufficiency appeared but this was
considered to be an unnecessarily severe test on the patient, and was abandoned.
Antero-posterior and lateral radiographs of the skull rvere taken before operation.
The anaesthetic was administered by a cuffed endotracheal tube passed through
the mouth. The nose was packed before operation with gauze soaked in cocaine
and adrenalin. The rods of either i^^Au or were inserted through a cannula
introduced into each nostril in turn. The position of the cannula wls controlled
by visualization on an image intensifier, working in turn planes.

Postoperative

The patient was nursed in a horizontal position for 12 hours and then allowed
to sit up. Cortisone was continued indefinitely and thiToid evtnet
when signs of thjToid deficiency appeared two to three months after operation"

630

W. P. GEEENING ET AL.

Complications

All patients developed headaclie but this was not usually severe and was
easily controlled by simple ajialgesics. In a few cases, persistent diabetes insipidus
made it iiecessary to give either pitressin taimate in oil or pitressin snuff. Bleediiw
Avas a common occurrence but Avas not often severe, though tAvo cases required
blood transfusion. Excessive bleeding Avas usually confined to those patients
in Avhoni the base of the skull Avas grossly involved b}' metastatic tumour. Optic
atrophy occurred in 3 patients and must be assumed to be due to the implant,
but unfortunate!}' this could not be confirmed as post-mortem examinations n^ere
not earned out on these particular patients. In one case, hoAveA'er, blindness ivas
discoA'ered, at autops}', to liaA'e been due to a metastasis in the optic nerve. The
most common, disastrous complication Avas rhinorrhoea folloAved by meningitis
AA'hich caused 10 deaths. Cerebrospinal fluid rhinorrhoea and meningitis, either
separatel}', or together, occuri'ed in 21 patients of AA'hich 9 had ^®®Au and 12 had
®®Y. Six patients deA’eloped meningitis without previous rhinorrhoea and 5

i8t

XX O Q Sx

X Rhinorrheea
O Meningitis

Meningitis + Rhinorrheea

-T~I

Implant

Months

Fio, 2 . — Onset of rhinorrhoea and meningitis following implant.

of these died. A striking feature of this complication Avas the A'ariation in the
lapse of time after implant before its occurrence. The diagram (Fig. 2) illustrating
tliis includes tAVo patients suffering from a disease other than cancer of the breast.

Antibiotic coA'er, as long as the leakage persisted, AA^as the only ti'eatroent
emplo 3 'ed. Meningitis AA’as extremefy resistant to treatment and the clinical
course varied from acute fulminating to chrome. At post-mortem examination,
multiple adliesions AA'ith much fibrous exudate Avas commonly found and it Avas
not surprising, therefore, that antibiotics had little effect in these cases. There
are seA'eral possible causes of the cerebrospinal fluid leak. There Avas no cor
relation betAA'een the activity of the rods and the deA'elopment of the rhinorrhoea,
but there is no doubt that it occurs if rods are placed high up anteriorly. In t ns
position thej' lie just under the diaphragma sellae and may cause it to necrose
(Forrest, 1959, personal communication). Rliinorrhoea can, hoAVCA'er, occur
Avith properly placed rods and Forrest suggests that it is then dAie to over osage,
but this does not explain the immediate leak of cerebrospinal fluid which we
have noticed on several occasions on the introduction of the cannula lov
near the floor of the fossa. The cause, therefore, probably lies in abnorma
anatomy and there are tAA'o A'ariations from the normal which may be impor an .
Firstly, the depth of the fossa, as shoAvn radiologieally, does not necessOT }
indicate the size of the gland which it contains (Fig. 3). The pitm ary 3

PITUIT.^BY IRKABIATIOX FOK HIUCAST CANCMT.

r,:i I

sometimes consist of no more <liiw flnwi.

and in sneh a case, the diaphragma is no . tln'rofore,

to he closely applied to the ,.f .pe

Mciemy is qiu'te large and it is in sncU cases thal Hie s„l,aracl,n..„l s,,:,-

extend down into the fossa.

SesuUs . ,

The assessment of the results of hypoiihyscctomy is (hnumlt In many
cases the disease is apparently arrested-no new lesions .niipeanng. hut without
any signs of healing of the original ones. In some patients marked sul)jeeliv(>
improvement occurred with no evidence of any objective remission. It iwis
decided, therefore, only to assess those cases who have shown definite objective
remissions hut tliis does not include “ arre.st of the di.sea.se. Many jiatient‘>
die from their disease 11111110 a short time of operation and the.se cannot, thendore.
be assessed but in order to give an accurate picture of the scries of 1 0(1 cases I hey
have been included, and no case has been removed from the series on the ground.s
that it has been impossible to evaluate the patient, either bceausc they have been
lost to follow-up, or, because they died too soon. All complications are included
and any death in which it is doubtful whether it was the result of the ojicratioii.
has, nevertheless, been included in the series. In addition to this, no patient
was refused treatment, either because they were unfit for operation, or because
it was considered that the disease was too advanced. The results, tlicrcfon-.
must be considered to be as complete as possible.

Only two patients are alive, although twelve (11— ’“^Au. 1— »'’Y) had objective
evidence of regression and one of these survives 44 montlis after imjilant. ’I'lie
remainder hved from periods ranging from 7 to 40 months, ivith an avenii:e
survival of 19-4 months. The sundval of the 88 patients who did not rt's-pond
IS shown (Fig 5) ; 65 were dead in six months and 18 lived for le.vs than one

month. The few who hved for relatively long periods remind one how chronic
this disease can be.

pituitari- implantation performed as the first i)Ianncti

pituitarj’ implantation failed to induce ®“'=pessful oophorectomy, and

reasonable comparison can be made of the re is obvious that no

by other methods of treatmenr tho.se obtained

Invesligaiions

^ foltowing

the

632

W. P. GREENING ET AL.

1. Urinary godadotrophin excretion.

2. Urinary oestrogen excretion.

3. Serum 17-ketosteroids.

4. Radioactive iodine uptake.

The urinary gonadotrophin and oestrogen assays were undertaken primarily
in order to see whether there was any correlation between the preoperative
levels and the results of treatment. It was also hoped to show whether there
had been complete pituitary destruction by comparing the postoperative findings
with those carried out before operation.

Fig, 5 , — Survival after implant (non-responders) = 88 patients.

It is not the purpose of this paper to analyse in detail the results of these
investigations, but some important points are noted. The estimation of gonado-
trophin excretion in the urine is the only satisfactory test available at present for
the measurement of pituitary function. The levels in premenopausal women
tend to be low. They rise after the menopause and the highest figures are usually
seen following castration. In anj'^ patient, however, whether premenopausal or
postmenopausal, there are wide variations from day to day and some solitary
tests are probably, therefore, valueless (Fig. 6). A fall to zero from a high value
before pituitary destruction indicates a considerable diminution of pituitary
function, but apaid; from a suggestion in those patients with moderately high levels
of gonadotrophin excretion before operation that a good result is probable, we
have not obtained any help from this estimation. The levels of oestrogen m

PITUrr.VRY IRRADIATION- ROR DUV-AST C'ANCV.I’.

fte .m.,e a» subject to «V Xm”'.:

Fig. G. — ^Post-menopausal woman, aged .GS.

Pathology

The extent of destruction of the pituitary was estimated in :UI specimens
obtained at post-mortem and examined histologically. In 4 the eland was

V'" r'

SM;, *1™ il:::

in most cases. In common ^^dth other workerf'^rV'' necro.si.s

not efficient in producing total nLosis anil J ’

10 ™l 20 pc, cent of a|pa,ent“'SLt!ir

634

W. P. GREENING NP AL.

levels fell to zero in both cases. Among the patients who did not respond there
were 6 in which the extent of destruction was between 80 and 100 per cent and
in all these the gonadotrophin excretion fell to very low levels after implantation.
In the other 2 cases the amount of pituitar}" destroyed was small, 10 per cent
in one and 33 per cent in the other, yet, there was considerable fall in the gonado-
trophin excretion. One must, therefore, accept noth caution a fail in nrinarj-
gonadotrophin excretion as evidence of complete histological destruction of the
pituitary, although it probably reflects loss of function.

Dismssion

Since there is considerable difference in the published figures given, both for
surgical hj^oph 3 'sectom 5 '’ and irradiation hj^poplij'sectoinj’', the result in compar-
able series of patients from the point of view of the remission rate, mortality and
percentage of complications etc. are summarized in Table I. The number of
cases of breast cancer treated bj^ surgical removal of the ly'popl^'sis is 342. The
average mortalitj’’ is 7 per cent, the complication rate 15 per cent and the remissions
46 per cent. In 675 cases treated bj’- interstitial irradiation of the pituitary', the
mortality is 3 per cent, the complication rate 32 per cent and the average remission
is 26 per cent.

Table I. — Ejfecfs of Interstitial Irradiation in Breast Cancer

A’uinber

mortality

Complications

Authors

of cases

(%)

<%)

Frnser et al., 1959

Ironside et al., 1959 .

—

. 77

Greening ct al. (this paper) .

100

Forrest ct a(., 1959

Bnuer, 19.50

400

(less than)

Total

075

Surgical

removal

Rny and Pearson, 1959

109

Kennedy et nl., 1956 .

Luftet al.. 195S

Bndley-Smith, 1900 .

Atkins el al., 1900

Total

343

Beneficial

response

(opprox.)

(arrest 9%)
53
26

(arrest 1-1%)
42

(arrest 10%)
GO

Cerebrospinal fluid leakage is the main problem to be solved in order to ma
interstitial implantation of the pituitary safe. Since the leakage usua y
tvhen rods are placed lugh up in the fossa and rarety when thej’^ lie on le i
high placement of the rods must be avoided and in order to do so the fossa ®
be approached horizontal^. A horizontal approach via the nose is •

in many cases and septal deviation and enlarged turbinates add to he i ^ ,
of the operation. We have therefore changed over to the transetlrmoi a
used by Bauer (1956), and a trial of tins method is being undertaken.

PITVITAKY TRKAIMATIOX I'OU ( AN( LU

We also consklev tUat^ J}|ort"m„ge luul vaii'l f*''! <'«'•

in all cases, it is preferable o u-c * ^ j . j Yin- lUiioiiiit ttf tfuliii-

and irill deliver an adequate close to all parts on lit ^ ^

tion required is not knoivii ivith any degree of certauitx but lor puri
proposed trial we are using 50 me of '-^Au.

Summary . .

Between rebruaiA^ 1955 and March 1 or, S. interstitial irradiation of the intuit ary

was carried out in 100 cases, using cither >»«Au or '•“Y. When all t rentetl pat i-nt*^
are included in the series and the strictest possilile enteria used the remn-ion
rate was 12 per cent, the mortality 11 per cent and the eompliealion rate I’l
percent. The patients treated were those for whom no other method of Ireattneiit
was available.

The operation itself is technically .simple ami in the majority nf e.tM-^. m,
more of an ordeal than ovariectomy.

It is suggested that this method of treatment is, in certain ca^es. well north
continuing and that its usefulness could he increased cousiderahly if the eonipli-
cation of rliinorrhoea were eliminated. Although it was originally nnderlahi'n
only in advanced cases of breast cancer, it has other s),hercs of nsefnlnes'* and
may be helpful both in acromegaly and pituitary tumours.

A further trial of the method is being unciertaken at juvsent. usini; '’‘Aii
grains implanted via the transethmoidal route.

We nish to thank all those authors who made their results availahh
Our thanks are also due to the Departments of Medical Art ami Phot
of the Eoyal Marsden Hospital.

Q ^ (Eamsey, 1960) are reproduced by ])ermission of the Eovnl

Society of Medicine.

to us,
ograptiy

Atkius, H. J. B., Falcoxeh, M. A. Hayward T T i- o l.

A^n ^mxAGE, P.-(i960) IRS ’ ''

Bauer, K. H^(1956) Arch. Min. Chir., 284, 43S.

'"'"''Toy, A. firK^yM,T, H,;u:s J.. Sandi-

Bnf. J. Surg., 47, 61. 'Ilemtye, J. M. axd Garter. P. T.~-{ 1

Mem, Peebles BHomc, D. A ^Iopris t

lancet, i, 399. ’ ' ’ iLLrvGwouTH. G. \V.~{i;),-,(i)

PRASER, B.. JoPLUC, G. P Law«; T W a\

^ Mid., i, 382. ■’ •’ R. and Stei.yer, R. K --(Ptyn

Hremvs T’ Brit. .J. Surg., 26 81 1

ni.GCriNs. L. A^^D Scott W w nn,4-v'^7 ’

636

W. P. GREENING ET Ah.

Lodge, W. C. — (1936) Brit. Med. J., ii, 1257.

Lett, R.. and Oltveceona, H. — (1952) Nord. med., 57, 351.

lidem, Ikkos, D., Nilsson, L. B. and Mossberg, H. — (1958) ‘Endocrine Aspects of
Breast Cancer ’, edited by A. R. Currie. Edinburgh (Livingstone), p. 27.
SIahmoud, M. E. S. — (1958) Brit. J. Radiol., Suppl. 8.

Noethmeld, D. W. C. — (1949) Proc. R. Soc. Med., 42, 845.

RADLEY-SmTH, E. J. — (January, 1960) Personal communication.

Ramsay, G. S. — (1960) Proc. R. Soc. Med., 53, 641.

Rasmussen, T., Haeper, P. V. and KjENNEDY, T. — (1953) Surg. Forum, 4, 681.

Ray, B. S. and Pearson, 0. H. — (1959) Cancer, 12, 1.

Tobias, C. A., LA^VHENCE, J. H., Born, J. L., McKombs, R. K., Roberts, J. E., Anger,
H. 0., Low-Beer, B. V. A. and Huggins, C. B. — (1958) Cancer Res., 18, 121.
Westminster Hospital Report. — (1956) Rep. Brit. Emp. Cancer Gampgn, 34, 173.
Ydhl, E. T., Harper, P. V., Rasmussen, T. B. and Bergenstal, D. M. — (1955)
Siirg. Forum, 6, 489.

THlf wmrTS OF 4- SENECIO ALKALOID (JIONOC'HOTALIKE)
™ oSlAN EMBRYO LIVER IN TISSUE CULTURE

VIVIENNE HIRCHINSON a>-u K. R. HILL
From the Pathology Department, Royal Free Hospital. Gray's Inn Roa-l. Lominn. 11 /. .I

Received for piiblicnlion August 23. lOfiO

This communication describes the effects of monocrotalinc. a jjvrrolizidine
alkaloid, on human liver cells in tissue culture.

Seneciosis is a liver disease which is due to the ingestion of certain jilants.
mostly of the genus Senecio, containing alkaloids of the pyrrolizidine group and
this condition has been a subject for research in this department for some time.
Many workers, including Schoental and Head (19.55), Bras and Hill (IbuG). Bcrr_\
and Bras (1957), Schoental and Magee (1959) and Hill (1959), Inave described
the various lesions produced both naturally and experimentall}', but the patho-
genesis of this disease is stiU obscure. Associated with other phenomena, several
authors have obsen'ed enlarged parenchymal cells with single hj'pertrophicd
nuclei in the livers of affected animals (Harris, Anderson and Chen, 1942 ; Hill.
1959 ; Stephenson, unpublished). Bull (1955) has termed this condition “ incgalo-
cjdosis ”.

Neoplastic changes have been reported in rat livers after injection with
pyrrolizidine alkaloids by Cook, Dufiy' and Schoental (1950) and Schoental, Head
and Peacock (1954) and in fowls by Campbell (1956). An investigation at the
cellular level, uncomplicated by vascular or other effects, was therefore thought
to be worth investigating.

JIATERIALS AHD 5IETHODS

A strain of human embrym liver cells (HuLi)* originallj' established by H^est-
wood, McPherson and Titmuss in 1957 was used for most of these studies. One
series of experiments was also performed with a strain of HeLa cells maintained
for some time in this laboratorJ^

All the cells were cultured in a medium which contained the following ingredients:
Calf serum (deactivated at 60° C. for 30 min.) . . 1-5 -0%

Lactalbumen hydrolj’sate
Yeast extract
Tryptic meat broth
Peptic digest of sheep’s blood
Earle’s saline to 100.

0-5%

5-0%

0-1%

Antibiotics were added in concentrations of •
penicillin, 100 units /ml.
mycostatin, 200 units /ml.
neomycin, 0-2 mg/ml.
streptomycin, 20 units /ml.

UMnined througli the kindness of
" ich General Hospital, Birmingham, !1.

• -Tiewett, Kegional '

5 x^aporj

Little Brom-

638

Vn^IENNE HIRCHINSON AND K. B. HILL

The medium was made up weekl}^ from stock solutions which were discarded
after six months and the fresJdj' dissolved antibiotics were added just before use.

Stock cultures were grown on glass in pyrex feeding bottles incubated at
C. and re-fed weekl 3 L Cells were removed from the glass by incubating for
5 min. Avitli O-l per cent trypsin (Difco 1 : 250) in pliosphate-buffered saline at
pH 7-2. The cell suspension thus obtained was spun at 1000 r.p.m. for 5 min.
After the supernatant liquid rvas removed, the cells were resuspended in fresh
medium by stirring magnetically for 5 to 10 min., then dispensed into fresh bottles.
Stock bottles received aliquots of 10 ml. with cells in the concentration of 2 x 10*
cells/ml.

The experimental vessels were Sanders PM/3 vials with silicone rubber-lhicd
screw caps, each containing a coverslip on whicli the cells settled. Two millilitres
of the cell suspension together with the substance under test was seeded into each
vial and incubated at 37° C. Tests were made with monocrotaline HCl, sterilized
by Seitz filtration, in concentrations of 1, 2-5, 26, 50, 125, 250 and 500 fig./ml
Other hepatotoxie agents, used in the following concentrations, for comparison
with monocrotaline, were carbon tetrachloride 0-01, 0-015, and 0-02 /il/in]. ;
thioacetamide 1, 2 and 4 /rg./ml. ; retrorsine 100 and 200 /ig./ml.; ethionine
100, 200, 500 and 1000 /ig./ml. ; 2:4 dinitrophenol 500 and 1000 /ig.jm).;
dimethjdaminoazobenzene 1000 /tg.jml. (Table I).

Table I. — Summary of the Treatments Accorded to Varmis Series of Test Cidtures

Series

Drug*

/(g./ml.

Solvent

Time of sampling

H63/1

, Monocrotaline HC'I

2-5, 25, 60, 125

deionised

HjO

, 1, 2, 3 days

126, 250, 500

, 3 and 5 days

250

. 3 to 7 days

1-0

. weekly for 15 weeks

»»

500

,, „ ® >>

Carbon tetrachloride

tO-01, 0-015, 0-02 .

Earles

saline

. 3 to 7 days

Tiiioacetamide

1,3,4

deionised

HoO

•

Retrorsine

100. 200

•

Ethionine

100, 200, 500, 1000 .

. weekly for 5 weeks

1000

2 : i dinitrophenol

500, 1000

absolute

ethnnol

. 3 to i days

. Dimethylaminoazobenzene.

1000

”

* These substances were added in a volume of OT mb of solution per 10 ml. of me^ura,

exception of carbon tetracliloride which
of 0-2, 0-3, and 0-4 ml. respectively,
t Concentration measured in /il./ml.

was added as a saturated solution in

Earle s sahne

In the case of tlu-ee long term experiments (5-15 weeks), the cultures veie
re-fed weeldy with medium containing a fresh inoculation of monocrotaline lor
experiments H63/4 and H63/5 and ethionine for H63/9. Subcultures were ma
when overgroudh demanded, using the same trjqisinizing procedure as betore ana
the controls were ahvaj^s subcultured at the same time. ^ i a

Changes in the appearance of the cells are well known in long-estabhs le
cultures and, in order to minimize errors in interpretation arising ®
alterations, each set of experimental cultures was paired rrith a set ol contempor i j

controls.

EYPECTS or MOXOCKOTALINE OX ElVVAl 'riSSL'K

The coverslips ,vit,h the adhcrh.g

control cultwTes each day from t le “ j ^ second and lllird day ami

?Sd"SftSa.oa“in i penodic acid-SchilT. and fornmlin lixed

used ou liver cells grown for a long time n. rWro. cultures of Ikd.a
treated for comparison, in the same manner as the liver cultures of sene.'-
(Table I) and incubated with -200 /(g./ml. of monocrotalino.

Mitotic counts

Normal and abnormal mitoses were counted for each day in cultures of fjeneJ’
H63/2 and 3. In each case 1000 cells were observed and the number of uutolie,
figures recorded.

Mnclmr measurements

The nuclei of haematoxylin and eosin stained cells of scries 1103/1 and 2,
receiving concentrations of 50 and 125 /ig./ml. of monocrotnliuc were mea.sured.
The slide was projected on to paper at a fixed magnification and a total of 250
randomly selected nuclei from at least 10 different fields was outlined. The
average of the greatest and smallest diameters measured at right angles was
calculated for each nucleus. It was not possible to do this for nuclei of cells of
cultures receiving higher doses of monocrotaline because of their irregular slinjie
and the occurrence of the bizarre forms to be described later in this paper.

RESULTS AXD OBSERVATION’S

Monocrotaline treated cidtures

For the first three days of culture and uith concentrations of up to 25 /ig./iul.
of monocrotaline, no visible differences could be detected between exjicriinenfal
and control cultures. At higher concentrations changes which increased in decree
u ith dose and length of exposure could be observed

These changes were ^t manife.sted as a slight but significant increa.se in
nuclear size (P _ 0-02) m the three day cultures receiving 50 fig.lml. of mono-
crotalme Tins can be demonstrated in the shift to right of averfge diameter "In
compared mth controls. Although cj-toplasmic area ivas not esti-
mated, the cytoplasmic-nuclear ratio did not annear tn Iny-o nUo i
a similar hjTiertrophy of this part of the lrisSr nlaraeme

arranged squamous cells became increasindv disnmf f "f evenly

appeared to be less cohesion between indhufinlT ? irregular and there
regular cell sheet (Fig. 4, 7), at three days the teut'^^ ff norniallv

groups and isolated cells with many m^nltk fnVlJ of scattered

cell clehns (Fig. .5, 6, 8 9 ) Therp i ^ individuals and much

»»ilyl,cK.g„„.l3h.peWthe cells

' 6 - ; 10, 20) to a number

640

VIVIENNE HIRCHINSON AND K. R, HILL

of mixed and varied forms. These included some spindle-shaped cells often m'tli
eccentric nuclei (Fig. 5, 6, 8, 9, 19), and others with very long attenuated crto-
plasmic processes which in some cases appeared to bridge two cells (Fig. 16)
Sucli piooesses were cspeeialiy prominent in older cultures with high doses of
monocrotaline where the cell population was very sparse. Abnormal hj^oertrophic
cells of a very bizarre aspect appeared in great numbers and were of two types :

3o[- l2S^g./nil.

2:1

Avenge dianncter (;j)

■ 1 *

I ’

I' i/ !i:

Fxg. 1. — Distribution of average nuclear diameters after tiiree days of incubation cultures of
HuLi cells receiving 50 or 1 25 ftg./ml. of monocrotaline. i^Ieasurements were made
in each case. There is a significant shift to the right in the test cultures P=0'02 and <0
respectively) indicating nuclear hj^jertrophy.

(a) giant cells with numerous nuclei wliicli varied considerably in size, shape and
number (Fig. 5, 8, 9, 13, 17, 18, 21), and (6) enlarged cells with a single huge
nucleus (Fig. 5, 6, 8, 9, 25), Giant cells of type (a) were especially numerous (big.
2), but both types of cell were very striking when compared with the ce s o
controls with their ovoid and rather regular nuclei and small well defined nuc eo i
(cf. figures of control and experimental cultures). .

Irregular, fused and misshapen nucleoli (Fig. 13, 14, 18, 25) were a fea ure

EFFECTS OF ^lOXOCROTALTXE ON LIVEU TISSl'E

the nuclei of both Idnds of enlarged cell in test cultures (we would like lo t<‘rin
these megalocjdes) and coarse, granular, deeper-staining cliroinatin was sonudiines
present (Fig. 5, 8, 14, 21, 25). The cytoplasm of the enlarged cells was often
tenuous or finely granular, and sometimes contained mimerous small or one or
two verj'^ large vacuoles which were Schiff negative and did not contain fat (h'ig.
11, 12, 17, 25). Amorphous eosinophilic bodies within vacuoles were sometimes
present in the cjdoplasm of haematoxylin and eosin stained tost cultures (I'ig. 1 1 .
16, 19) although these were verj' occasionally seen among cells of the cc)utrols.

Culture No.

Dotage Monocrotaline

3 5

H63/2

abnormal cells Th^ the appearannp nf ■^■^‘'‘^"lerease

of the test culture.,. ""»'««■ variability nS?reach Iho etc S tlmt

dfitoPc counts and growth rate

e ““'“a'l-We apace becaiS^tSi^g'

VIVIENNE HIRCHINSON AND K. R. HILL

The picture presented by the test cultures was more erratic. The number of
mitoses did not diminish as expected. On further examination (Fig. 3) a large
number of divisions— in some cases nearly 60 per cent of the total-appeared
abnormal m some way, with at3TjicaI spindles which were often multipdar or
defornied. Heteroploidy was common and sticky or clumped chromosomes often
seen (Fig 14, 16, 22, 23, 24). Abnormal mitoses were rarely found in controls
(fig. 3). Some cells of test cultures appeared to be dividing but without evidence

Experimental cultures receivlnj Monocrotaline
Dosage 250 ug/ml.

Culture No. H63/3

SOO ug/ml. 250ug/ml.

H63/2

Fjg. 3.— Tottil, normal and abnormal mitoses per 1000 cells in HuLi cultures receiving 250
or 500 pg./nil. of monocrotaline. The height of each column represents the total mitoses,
while normal and abnormal divisions are shosvn by the tmshaded and crosshatched areas
respectively.

of a mitotic spindle (Fig. 13) and no mitosis was ever observed in a giant cell
although several of these had constrictions of the cjdoplasm (Fig. 13), c.vtoplasnuc
bridging (Fig. 16) and nuclear indentations (Fig. IS).

Long term experiments with monocrotaline

The experiments were performed to investigate the action of repeated high
and low doses of monocrotaline. Cultures of H63/4 receiving 1 yg./ml. per wee
were not visibly different from controls after 15 weeks. At this time the cu tures
were accidentally lost. In the second experiment H63/5 with the high dose o
monocrotaline of 500 yg./ml. per week, the bizarre changes rapidfy appeare ,

EFFECTS OF MOXOCROTALIXE OX LIVER TlSSnC

the cultures declined verj^ quickly and died. After eight wcclcs only a few jiyknol ie
cells remained clinging to the glass vessel.

Experiments idth other Iiepcitotoxic mjents

Of the substances, in the doses used {Tabic 1 ). only rolror-sine (anollicr
pjTTolizidine alkaloid), 2 : 4 dinitrophenol and diinethylaininoazobonzene jn-r)-
duced megalocjdes vhich resembled those induced by inonocrotaline. Uelror.^inc
appeared to affect HuLi to the same degree as inonocrotaline, but abnormal cells
Avere few in cultures receiving the other two substance.';. Mypertrophied mono-
nuclear cells Avere not especially obvious after treatment Avilh 2 : •) dinit rojihcnol
or dimethylaminoazobenzene.

Experiments with HeLa cells

As in controls of HuLi, control HeLa cultures also contained a few miiltinuclear
and enlarged cells, but the addition of monocrotalinc did not seem to be remarkable
m its effects.

tjTe have not been described in vivo

prrohzidine alkaloids or in the naturailv administration of

however, haA*e been obseiwed in many esLblished .^^"^^""'alcate cells,

non-mabgnant origin after long perLds in vUrn t malignant and

phieh possessed many nuclei in cultures from

I^lh, Balducci, Gon and Bondi (1957) noted “ imnian nasal mnco.s-a and
liver KB and HeLa cultures. Berman Sb "«nnal Imm";;

All Srutt,”"' "“"y bna'"' "'’»■) “IKO moiition

c^t ^ on controls Serried ag c s

::h~

Vn^IENNE HIRCHINSON AKD K. E. HILL

and also larger numbers of giant cells appeared in cultures of nine different strains
including normal human liver, after exposure to irradiation. Heteroploidv had
been demonstrated in these cultures and Pomerat et al. ( 1957 ) attribute multi
nuclear giant cell formation to this, suggesting that they are the result of several
mitoses ivitliin the same cell -where subsequent cytoplasmic division fails to occur.

EXPLAHATIOH OF PLATES

Fig. 4.— Tlireo dny control culture, showing sheet of regulor nnd rnther cohesive cells yrith
severnl mrtotic figures. Cf. Fig. 5 nnd 6. H. nnd E. x 85.

Fig. 5. Three dny culture ^rith 250 /rg./ml. of monocrotnline. Disrupted and less cohesive cell
sheet showing .slight increase in cell nnd nuclear size compared with control in Fig. 4. Xote
^ bizarre forms, spindle-shaped cells nnd mono and multinuclear giant cells. H. and E. X85.
Tig. 6. Three day culture with 500 //g./ml. of monocrotnline. As Fig. 5 but showing more
exaggerated effect. H. nnd E. x 85.

Fig. 7. — Five dny control culture. Squamously arranged dense sheet of cells covering most of
available space with onlr’ sliglit variations in cell size. H. nnd E. xS5.
h IG. 8. — Fiv'C dny culture with 250 //g./ml. of monocrotnline. Poorly populated culture with an
increased proportion of biznrre cells niien compared with Fig. 5 and 6. Kote fine cj-toplasmic
extensions nnd frequent vncuointion. H. nnd E. x85.

Fig. 9. — Five dny culture with 500 //g./ml. of monoorotoline. As Fig. 8. H. and E. xS5.
Fig. 10. — Three day control culture, for comparison with Fig. 11-19. H. and E. x340.

Fig. 11. — ^Three day culture with 250 //g./ml. of monocrotnline. Binucicnte cell containing an
eosinophilic body within a vncuolo {->•). An adjacent multinuclear cell has finely vacuolated
cj'toplnsm. H. and E. x340.

Fig. 12. — Three dny culture with 250 //g./ml. of monocrotnline. The centre cell contains one
huge vncuolo which hns pushed aside the nuclei. H. nnd E. x 340.

Fig. 13 . — Tlirce dny cultures with 250 //g./ml. of monocrotnline. A multinucleated cell with
a central constriction (— >) suggesting amitotic division. Xote the irregularly shaped nuoleoli
in surrounding cells, H. nnd E. x 340.

Fig. 14. — Three dny culture with 250 //g./ml. of monocrotnline. Many nuclei show fused
nucleoli including the nucleus (a) which also contains hjqierchromntie material concentrated
at the edges of the nuclear membrane, (b) A quadripolnr mitosis nearing completion.
H. andE. X340.

Fig. 15. — Three day culture with 500 /.g./ml. of monocrotnline. Group of abnormal mitoses
shom'ng “ sticky ” and clumped chromosomes. H. and E. x 340.

Fig. 1G. — Three day culture with 500 //g./ml. of monocrotnline. Bridging of cj-toplasm (->)
between two cells. Note eosinophilic bodies. H. nnd E. x340.

Fig. 17. — Three day culture nith 500 fig. /ml. of monocrotnline. An enlarged binucleated cel!

with vacuolated cytoplasm. Cf. size of the nuclei with controls in Fig. 10. H. andE. x340.
Fig. is. — T hree daj' culture witli 500 fig. /ml. of monocrotnline. TSvo multinucleated giant cells.
Cell (a) hj-perchromatic nuclear material, irregular nucleoli and an indented nucleus (-?■)•
Cell (6) a large number of extremely small nuclei. H. nnd E. x 340.

Fig. 19. — Three dny culture with 500 //g./ml. of monocrotnline. Spindle-shaped cells witli
eccentric nuclei. H. and E. X 340. , ,

Fig. 20. — Five day control culture for comparison with Fig. 21-25. Small cells of equal size witn
ovoid nuclei and small nuoleoli. H. nnd E. X 340.

Pig. 21. — Five day culture with 250 //g./ml. of monocrotaline. Giant cell with numerous nuclei
of varying size. Note darker staining nuclei of surrounding cells. H. and E. X 340.

Fig. 22. — Five dav culture with 250 fig./ml. of monocrotaline, Qundripolar mitosis in me a-
phnse. An adjacent cell also shows abnormal mitosis. H. and E. x 340.

Fig. 23. — Five day culture with 250 //g./ml. of monocrotaline. An enlarged mononuclear ceii
in abnormal mitosis at an earlier stage (raetaphase) than that of Fig. 24. The mononuc ear
origin of the chromosomes is obvious here. H. and E. X340.

Fig. 24. — Five day culture with 250 //g./ml. of monocrotnline. (a) Greatly h^’pertropme

nuclear cell showing abnormally high number of chromosomes with five mam condensa lo .
Scattered, isolated chromosomes can also be seen, |b) Cell in late telophase, (cj
metaphase, note misplaced chromosome. H. and E. X 340. .

Fig. 25.— Five day culture with 250 //g./ml. of monocrotaline. Mononuclear “T „•

hjqierchroraatic nuclear material and abnormal nucleoli. Cf. size of cells oi con r
20. H. and E. x 340.

BlilTISH JOLT.VAL OF CaN'< F!!.

V.il. MV. S'K I.

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Hircliiiisoii nnd i-fj||

British Joitrn'al or Canter.

Vol. NIV. N... 1.

nmi Hi||,

British JoirjiN'Ai. ok Canckr.

V..! t

Hirchinson mi,i nj||

uir.

eptects oe MOEOC1U.TAUSE fS i.'Vi'.r. TlEM-i:

Hete, opioid), .ud many nndHpol.v milo.ic llenro, no,o fr,.„.«.nlly .d'

mdri;

Bucher (1958) u’ho sudi n CDmlilii'H uro'-'- hv nttiil-ti'

osteoblasts and fibroblasts and cone ItinnHrnt.’ (and ib-ur •

division. According to tins author . - ; „f ainitolic <livih..!i uliirb

-oiino,...,,;...,

cultures. In favour of the existence of nmit-osis m our nillure^.. b fni ..m
mitoses vere never observed in nniltinneleate giant relN or m lb- nnni.-r.nn.
binucleate cells, while there were often constrictions of tbe cylo].biMn and ind-Mta.
tion and budding of the nuclei, suggesting that some sort of division no*- in i*ro.
gress While some multinuclear giant cells may have arisen by iiiiI.'m** in tlm
fashion described by Pomerat el al. (1957), it is iirobable that ninitotie diviM-.n
occurred in established multinuclear cells.

Another factor in favour of amitotic activity, is that (he tot.al miinber oftniio'-r,
of test cultures compared with controls, did not dilTer greatly except towiird ih"
seventh day of culture. The reason for this dilTcrcncc may he that at thi'- tiiii” the
cells of the control cultures had colomzcd most of the available sjiaee and growth
had slowed to a minimum maintenance rate, hut in test enitnres there wr\>- iinieh
cell death leaving a considerable area of impopnlatcd suhstratiiin. 1 f t liis explana-
tion of the minor difference in rate of mito.ses is acceptable, ninitotie aelivity
must have occurred in what would normally he the resting cells at the tiiiio of
sampling to produce the enormous numbers of multinuclear giant cells. Mono-
crotaline (and retrorsine) thus can he assumed to stimulate amitotic division.

The characteristics of the cells described in experimental test cultures such as
increase in size, pleomorphism, the great variation in nuclear size, shaiie ami
number per cell, the irregularities of the nucleoli and the coanseness of llie
chromatin matenal are features which frequently have been nscrilied to iieoiibmtie
cells. M bile such changes were not entirely absent in the controls and Imve often
been desenhed m long established cell lines, the marked rapid incre.ase in these
features after exposure to monocrotaline may be stressed A similn i- inr-m . ■

prepared animal and human hosts. These Lthal f Miitulily

a histologically mabgnant annearance rlpvpl i paljiahlc nodule.s of

which

, P»d»»drcrsr„;z;; "T„ r?" y -no™;,;,;:

«)«t monooroWine is ramawf of ><■ « possible therefore

;e at least i?;

iiuv inonocrotaline is canaWp pf possii

'■'■'r^in a tissue of normS origin. ^ ^ ^ neoplastic cliang,

Finally it should be added that riip fi i-
^vlth reservation when anolipff.p P>^esented must he interpreted

ions .1

of.an e.stablished slTinSls'

original state.
47

Buch as HuLi "inst'lSy rve^'lter^^^

646

VIVIENNE HIRCHINSON AND K. K. HILL

SVmtASV

1. A strain of human embryo liver cells was used to investigate the action of

the pyrrolizidine alkaloid, monocrotaline, other known hepatotoxic agents beine
used for comparison. ^

2. A strain of HeLa cells was treated with monocrotaline for comparison with
liver cells.

3. The first effect of monocrotaline on liver cells, noted at three days, with
doses of 50 and 125 //g./ml., was a significant increase in nuclear size. With
higher doses of the drug, cells became less cohesive and increasing numbers of
bizarre cells appeared. The latter included two types of megalocyte : (a) mono-
nucleated and (6) multinucleated cells. The multinucleated cells contained
varying numbers of unequal nuclei and comprised about 3-13 per cent of the total
population compared unth only 0-0-2 per cent in controls.

4. Abnormal mitoses were much more frequent in test cultures.

5. There was some evidence to show that amitotic divisions occurred in mono-
crotaline treated cultures.

6. Teatures similar to those seen in neoplastic cells were also seen to increase
in cultures receiving monocrotaline.

7. Of the other drugs used, only retrorsine, 2 : 4 dinitrophenol and dimethyl-
aminoazobenzene produced changes similar to those described for monocrotaline.

8. HeLa cells remained unaffected by monocrotaline.

9. The findings suggest that monocrotaline induces in vitro, amitotic division
and possibly neoplastic changes in embryonic liver cells.

The authors would like to thank the British Empire Cancer Campaign for the
generous grant-in-aid which made tliis work possible and to acknowledge the help
of Miss A. Demery, M.A., Miss H. Haestier, Miss U. Hopkins, Mrs. A. Birbeck
and Mr. B. R. Phillips in the preparation of this paper.

REFERENCES

BERJtAN, L., Stulbeeg, C. S. and Ruddle, F. H. — (1957) Cancer Res., 17, 668.
Bebky, D. M. and Bras, G. — (1957) rV. Amer. Yet., 38, 323.

Bras, G, and Hill, K. R. — (1956) Lancet, i, 960.

Bucher, 0. — (1958) Z. mikr.-anat. Forsch., 64, 174.

Bull, L. B.— (1955) Aust. vet. J., 31, 33.

Campbell, J. G. — (1956) Proc. Roy. Soc. Edinb. B., 66, 111.

Cook, J. W., Duefv, E. and Schoental, R.— (1950) Brit. J. Cancer, 4, 405.

Harris, P. N., Anderson, R. C. and Chen, K. K.— (1942) J. Pharmacol, 75, 83.
Hill, K. R.— (1959) Trans. R. Soc. trap. Med. Hyg., 53, 217.

Idem AND Mahitn, H. M. — (1958) Brit. vet. J., 114, 345.

Jordan, W.S.—(1956)Proc. £!oc,e.rp. Biol. AC Y., 92, 867. c- •/ ?n

Belli, G., Balducci, D., Gobi, G. B. and Bondi, M.— (1957) R.C. 1st. sup. Sand.,

1113

Moore, A. E., Southam, G. M. and Steinberg, S. S. — (1956) 'S'cmnce, 124, 127.
POMERAT, C. M., Kent, S. P. and Logie, L. C. — (1957) Z. Zelljorsch., 47, 158.
Schoental, R. and Head, M. A. — (1955) Brit. J . Cancer, 9, 229.

Idem, Head, M. A. and Peacock, P. R. — (1954) Ibid., 8, 458.

/dem AND hlAGEB, P. N. — (1959) Acta Un. ini. Gancr., 15,212^ nr\Ka\ Ttrif 1 exv
Westivood, j. C. N., McPherson, I. A. and Tit»iuss, D. H. J.— (1957) urn. j. n

Path. 38, 138.

;=;■ ■> »•'“

Received for publicnlion Seplcml)cr 10. lOf.n

ol eTen norntS (Hc^J^r li"r> : Tnuil.^upl KH.
1940; Webster, ef a/., 1944; Willis, 194S). Other workers hmNTyrr (Slnup^^^^^

1948 ■ Davis. 1955 ; Pack, 1957 ; Belisario. 1959) do not accept this thens lhr\
believe that as yet there is no authenticated case of a mole having become malignant
as a result of trauma and that the local appearance of a malignant melanoma alter
incomplete removal of a supposed benign naeviis indicates tliat the lesion na-
already malignant before removal.

A parallel experimental investigation of this problem is difliciilt. for nntnr.illy
occurring melanotic naevi are not to be found in common laboratory animals.
However, numerous melanotic tumours are readily produced by tlie re))e.ate<l
application of 9 ; lO-dimethyl-l : 2 benzanthracene (DM BA) to liamster .«):in
(Della Porta et al., 1956 ; Gbadially, 19.59 ; Ghadially and Barker. 1960). 'I'ln*
majority of these are benign lesions and in that respect at least they are similar
to the common mole of man. It seemed intere-sting therefore to inve.stigate whid her
mechanical trauma could convert such benign melanotic tniiionr.s into nmlignant
ones. It is realised that such a study cannot directly bear upon the controversv
regarding the effect of trauma to human naevi, hut ncvcrtliclcss it is important
to find out if an experimental situation can be created in which trauma turns a
benign melanotic lesion into a malignant one.

JIATEEXALS AXD JIETHODS

Brown and vrbite hamsters weighing approximately 90 g. were painted once a
week on the left flank with an 0-2 per cent solution of DMB A in acetone for a iieriod

^ ^ approximately one month animals liaring

melanotic tumours were segregated. The tumour-bearing flank of everv aniimd

Experiment 1

mm. to 3 mm. ^Sldi^fd^d^nto f from

bearing 48 tumours between them anil control erLn of animals

of similar size and distribution. ^ P of 10 shomng 3,5 tumours,

646

VIVIENNE HIRCHINSON AND K. E. HILL

SUMMARY

1. A strain of liuman embryo liver cells was used to investigate the action of

the pyrrolizidine alkaloid, monocrotaline, other knoivn hepatotoxic at^ents being
used for comparison. ® ®

2. A strain of HeLa cells ivas treated vith monocrotaline for comparison irith
liver cells.

3. The first effect of monocrotaline on liver cells, noted at three da 3 ’-s, with
doses of 50 and 125 //g./ml., was a significant increase in nuclear size. With
higher doses of the drug, cells became less cohesive and increasing numbers of
bizarre cells appeared. The latter included two t3T5es of megaloc 3 de : (a) mono-
nucleated and (d) multinucleated cells. The multinucleated cells contained
varying numbers of unequal nuclei and comprised about 3-13 per cent of the total
population compared with only 0-0-2 per cent in controls.

4. Abnormal mitoses were much more frequent in test cultures.

5. There was some evidence to show that amitotic divisions occurred in mono-
crotaline treated cultures.

6. Features similar to those seen in neoplastic cells were also seen to increase
in cultures receiving monocrotaline.

7. Of the other drugs used, onl}’- retrorsine, 2 : 4 dinitrophenol and dimethyl-
aminoazobenzene produced changes similar to those described for monocrotaline.

8. HeLa cells remained unaffected by' monocrotaline.

9. The findings suggest that monocrotaline induces in vitro, amitotic division
and possibly’- neoplastic clianges in embrj'onic liver cells.

The authors would like to tlianlc the British Empire Cancer Campaign for the
generous grant-in-aid which made tlu's work possible and to acknowledge the help
of Jliss A. Demery, hl.A., hliss H. Haestier, Miss U. Hopkins, Jlrs. A. Birbeck
and Mr. R. R. Pliillips in the preparation of tliis paper.

REFERENCES

Berman, L., Stulberg, C. S. and Ruddle, F. H. — (1957) Cancer Res., 17, 668.
Berry, D. M. and Bras, G. — (1957) N. Amer. Vet., 38, 323.

Bras, G. and Hill, K. R. — (1956) Lancet, i, 960.

Bucher, 0. — (1958) Z. mikr.-anat. Forsch., 64, 174.

Bull, L. B.— (1955) Aust. vet. J., 31, 33.

Campbell, J. G. — (1956) Proc. Roy. Soc. Edinb. B., 66, 111.

Cook, J. W., Dufpy, E. and Schoental, R. — (1950) Brit. J. Cancer, 4, 405.

Harris, P. N., Anderson, R. C. and Chen, K. K— (1942) J. Pharmacol., 75. 83.

Hill, 1C. R.— (1959) Trans. R. Soc. trop. lied- Hyg., 53, 217.

Idem AND Martin, H. M. — (1958) Brit. vet. J., 114, 345.

Jordan, W. S. — (1956) Proc. Noc. e-rp. BioZ. ALT., 92, 867. cy •< ?n

Belli, G., Balducci, D., Gori, G. B. and Bondi, M.-(1957) R.C. 1st. sup. Saint.,
1113.

Moore, A. E., Southam, C. M. and Steinberg, S. S.— (1956) Science , iZi , 12 1 .
POMERAT, C. M., Kent, S. P. and Logie, L. C.— (1957) Z. Zelljorsch., il, 158.
Schoental, R. and Head, M. A. — (1955) Brit. J. Cancer, 9, 229.

Idem, Head, M. A. and Peacock, P. R. — (1954) Ibid., 8, 458.

/Jem AND Magee, P. N. — (IQbd) ActaVn.int.Cancr.,\i,^2. 7 exp.

Westwood, J. C. N., McPherson, I. A. and Titjiuss, D. H. J,— (UOC ^ ru . .
Path. 38, 138.

THE

conv
is

WFECT OF TEATOIA OX THE MELAXO'I'K- TUMOlHiS OF
EFFLti Oi aOISTER

F. K. GHADIALLY, 0. ILIAUX AM> J. F. EAltKKH
F,„ oj TaiMm a.i iU C«««r ,■! SH"'

Kcccivcd for publirntion Sc|)t<-.nWr I'.>, Hlf.O

Mechamcal trauma has frequently been nccuscd of producinp ’I''

iSFenign tumours iuto malignuot ones. A., o, .Island, ug ,.xn,„,,lr .. 1 ,s

.. the malgnant melanoma of man tvhicl, many Wievc : .f

trauma to a mole (naevus) or even normal skm (Hewer, \Xl> . i raul and Ki ).
1940 • Webster, et ah, WW ; Willis, 1948). Other workers however (Slau}: , <T.
1948 ’• Davis, 1955 ; Pack, 1957 ; Belisario, 1959) do not accept this the.'sis, I he\
believe that as yet there is no authenticated case of a mole baying become maligimnl
as a result of trauma and that the local appearance of a malignant melanoina after
incomplete removal of a supposed benign naevus indicate.s that the lesion was
already malignant before removal.

A parallel experimental investigation of this problem is didiciilt, for naturally
occurring melanotic naerd are not to be found in common laboratory anitnals.
However, numerous melanotic tumours are readily produced by tbc rejieated
application of 9 ; 10-dimethyl-l ; 2 benzanthracene (I)MBA) to hamster skin
(Della Porta et ah, 1956 ; Ghadially, 1959; Ghadially and Barker, HKUi). Tlie
majority of these are benign lesions and in that respect at least they are similar
to the common mole of man. It seemed interesting therefore to investigate whethfu
mechanical trauma could convert such benign melanotic tumours into malignant
ones. It is realised that such a study cannot directly bear upon the contrfiver.sy
regarding the effect of trauma to human nae\*i, but ncvertbele.ss it is inqiortant
to find out if an experimental situation can be created in which trauma turns a
benign melanotic lesion into a malignant one.

3UTEBTALS AKD METHODS

Brown and wdiite hamsters weighing approximately 90 g. were painted once a

If f solution of DMBA in acetone for a period

25 weeks. After a rest penod of approximately one month animals hearinc
melanotic tumours were segregated. The tumour-bearing flank of every animal
was photographed and a chart indicating the size and nnsitinn nf ? *
around the costo-vertehral spot prepared for future referJ^ace and for nWr
co„r.c of events. Three separate experi^nls nere perfonjed as foH «

Ex-pmmtnl 1

1 brown hamsters bearing melannl'Tn

mm. to 3 mm. were divided into two groups an si^ie from

bearing 48 tumours between them and a contS onn f
of similar size and distribution showing 35 tumours

648

F. JST. 6HADIALLY, 0. ILLJIAN AND J. F. BARKER

The 48 tumours on the experimental animals were traumatized by priokine
with a needle (0-5 mm. diameter) right through the tumour and the full thickness
of tlie skin. Witli the needle in this position the tumour was further traumatized
by squeezing it on to the needle with a pair of forceps. This procedure was repeated
at weekly intervals on 6 occasions. The animals were observed for a further period
of 4 months.

Experiment 2

Five brown hamsters bearing eleven tumours varying in size from | to 3 mm.
were traumatized by cutting across them with a pair of scissors. The cut extended
right through tlie skin into tlie subcutaneous tissues. This procedure was repeated
after a fortnight. Another 5 animals bearing 14 tumours served as controls. These
animals were observed for a further period of 3 months.

Experiment 3

Seven white and fiA'e brown hamsters bearing 30 melanotic and hypomelanotic
tumours ranging in size from 3 ram. to 14 mm. were available for study. (Smaller
tumours were also present in these animals but they u^ere ignored for the purpose
of this experiment). Each of these animals also bore confluent ulcerated kerato-
acanthomas or squamous carcinomas. These tumours were excised and the
margins of the wound everted so as to expose the under surface of the adjacent
melanotic tumours. The base of seven melanotic and hjqiomelanotic tumours
exposed in this fashion was shaved off so as to liverate the tumour from its en-
sheathing capsule and bring it into free contact with underlying tissues. Another
seven tumours were traumatized by placing a chrominized catgut ligature in their
substance and a further seven were damaged by incorporation into the margin of
the wound created by the excision of the epithelial tumours. Nine untraumatized
tumours on the same animals served as controls. These animals were observed
for a period of 4 months after the infliction of the trauma.

RESULTS

In none of the traumatized tumours did we observe any rapid increase in size
nor did they show obvious malignant transformation by infiltrating the deeper
tissues or producing metastases in distant organs. Indeed some of the small
tumours traumatized in Experiments 1 and 2 became paler and smaller and at
times disappeared completely from sight. This phenomenon was also seen to
occur occasionally in small untraumatized tumours in control animals but it was
more frequent in the traumatized tumours. Fig. 1 shows a traumatized tumour
wdiich appeared to become paler. There is an obvious paucity of pigmented

EXPLANATION OF PLATE

Fig. I. — Traumatized melanotic tumour showing paucity of pigmented tumour cells in the
superficial part of the tumour. H. and E. x 155.

Fig. 2 . — Melanotic tumour fragmented by trauma. H. and E. X255. ^ photograph).

Fig. 3. — Oedematous vascular scar in traumatized tumour (pale areas

Masson’s trichrome X 155. . , , ..nmer and

Fig. 4. — High power view from Fig. 3. Note oedematous collagen in top lelt- ’

blood vessels flanked by melanin-containing cells. H. and E. X 620.

British JorRKAi. or CAS'ri:i:.

V..! MV. N. I

MELAKOTIC tumours of the IIAMSTEU

nr.t

c.,s particularly in ti.e ™i-« 1';;;:^, !:!;r"Ki;:1

foci of inflammatory cells . ^Kcoim rrnpmi-nicil u» » rosiill of

:rl':r3 in!i„r

slions a largo liriio-molanotic tumour Iranninliarfl l.y ioMTlioii of »

ligature which later drlppcJ of- •' ' '"'u'li; r,"ri'l,"re me

tumour. This tumour is markedly hypomclnnotm >ot ■ ’

many coUections of cells containing abundant melanin (l ip •«)■ H ' ' ‘I • ' ^
that trauma has stimulated local pigment production and or f ' ' ' '

feature rvas obsen-ed in other Iniiomclanotic or virtually amclanoln tnniom>

also.

DISCUSSION"

111

Our results shou" that single or repeated mechanical trauma lia.s failed to efTeft
an indubitable malignant transformation of a melanotic tumour in the hamster.
This is in keeping "with current opinion that trauma does not precipitate malignant
change in human moles (Belisario, 1055)).

It is e.\tremely difficult to evaluate the role of trauma in tumour ])rodnetion
or in the transformation of benign tumours to malignant one.«. Species dilTen'iiei
seems to play a dominant role. Mechanical trauma to carcinogen -treated skii
of the rabbit is readily followed by the appearance of tninonrs. Init in the mon«
rarely can tumours he produced bj" this method (Bercnbliiin. I'.t") !).

In experiment 3 a large deep wound was created in each animal by the operat ive
technique involved. This did not lead to the production of any new epithelial or
melanotic tumours around the margins of the wounds, though a tvjie 111 kerato-
acamhoma situated near the margin of a wound seemed to sliow a Imiiporary
rapid increase in size. The situation in man is difficult to assess. Imt rojiorts of a
variety of cutaneous tumours occurring at the site of injury may he foiintl in the
literature (Belisario, 1959).

The increased production of melanin in injured hypomelanotic inclaiiomns i.s
0 some biological interest. It is well known that hyperpigmentation inny occur
around traumatic scars, particularly those produced by burns. Such hyperpig-
mentation is also seen around insect bites and after the application of carcinogenic

increased pigmentation seen in traumatized
of thp l"? melanomas is probably no more than a reflection of the propertv

"uen Situated m a traumatized area.

tmimaUz^d mdintuf traumatized and non-

pointed out occasionally disappear from sight. It niiisl

It ^uVu 1 null i n sSe

Pf^inting roLut sM^dt? epithelial tumours produced by

i^'^truereSSTr-r ? disappear. In tliis Sse ?t

«f •'» prtS S'L' It -I ‘t:„d

650

F. N. GHADIALLY, O. ILLMAN AKD J. F. BARKER

from sight by discharging its melanin rather than by a loss of cells. If that is
the ease it cannot be considered a true regression.

sroiaiAEY

Single or repeated trauma to carcinogen induced melanotic tumours of the
hamster did not produce malignant change in these tumours. In the majority of
cases these tumours were not materially' affected by trauma, but in some instances
small tumours disappeared from sight. In larger hypomelanotic tumours a
vascular scar was formed, surrounded by cells containing abundant melanin.

We are indebted to Mr. T. L. Platts and Miss S. D. Wall for photomicrographs,
to Mr. J. N. Carver and Miss J. A. Osborne for technical assistance. This work was
supported by' grants to one of us (F. N. G.) from the British Empire Cancer
Campaign and the Uiuversity of Sheffield Medical Eesearch Fund.

REFERENCES

Belisario, j. — ( 1959) ‘ Cancer of the skin London (Buttenvorths), p. 190 and p. 20.
Berenblum, I. — (1954) Cancer Ees., 14, 471.

Davis, J. — (1955) Postgrad. Med., 18, 138.

Della Porta G., Rabpaport, H., Saffiotti, V. and Shubik, P.— (1956) Arch. Path.,
61, 305.

Ghadially, F. N. — (1959) J. Path. Pact., 77, 277.

Idein AND Barker, J. F. — (1960) Ibid., 79, 263.

Hewer, T. F.— (1935) Ibid., 41, 473.

Pack, G. T. — (1957) Virginia med. (Semi-)Mon., 84, 111.

Slaughter, D. P. — (1948) Snrg. Clin. Ak Amer., 28, 69.

Traub, E. F. and ItoL, H. — (1940) Arch. Derm. Syph., Chicago, 41, 214.

Webster, J. P., Stevenson, T. W. and Stout, A. P.— (1944) Surg. Clin, h . Avm.,11,
319.

Willis, R. A.— (1948) ‘ Pathology of Tumours’, London (Butterworths), p. 906.

G. R. CLERIC) AND E. W.

Received for iniblicnt ion OcloiH'f 8. 111811

to eLs«te to the careinoeenic s,.b.,ta.;ce» F““' '""'''""'f 'V, ,„t;

cigarette smoke and city 0 ...,^..-. — ^

designed to test this hjTothesis. Since the cxpcn.nents wero imgun t hn c >
ago Gellhom (1958), Roe, Salaman and Cohen (19:)'.)) and Wyndcr and IlofTinan
(1960) have confirmed the presence of incomplete carcinogens in eigandte nnoUi'
as foreshadowed by Hamer and Woodhousc (lOaO) and Cwynn and Snlainaii
(1956).

MATERIAL A^'I> METHODS

One hundred and fifty-six G57BL mice (68 females, 88 males) were divided
into 7 groups for the purposes of treatment. Aged lictwccn 8 and 6 wei'ks at the
beginning of the experiment, the mice Avere allowed to live to the entl of their
normal lives or, in the case of those u'jth skin tumours, they were killed when
the tumours became large or frankly malignant. All .skin tumonr.s except the
smallest papillomata were sectioned and examined histologically. A tumour
was judged to be malignant when the panniculus carnosus was invaded, 'rumours
were classed as “ probably mabgnant ” w'hen the tumour cells had not yet reached
the muscle although other signs of malignancy were present.

The test substances in solution rvere applied with two strokes of a No. 4 jndnt-
brush to the skin in the interscapular region. There were tiiree sidjstancc.s :
ftaction “ C ” from city smoke, a knoAvn carcinogenic material (Clemo, Miller ami
lytaus, 1955), applied as a 1-0 per cent solution in benzene ; croton oil a known
umour promoter, applied as a 0-5 per cent solution in acetone ; and the iiculral
fraction of cigarette smoke applied as a 10 per cent solution in benzene Tlii.s
ast fraction was extracted from the whole tar from cigarette smoke as described
S and approximately 6-4 mg. was appfed to

Gro^mf3«■*^*^^ experiment received the following treatments •
for 2 weeks only, witli fraction “ cT^'^They Svlrno f ’ t

fraction 3 times a week till death. ^ painted with neutral

652

G. B. CLEMO AKD E. W. MILLEB

Group III.—Twenty-four mice (15 females, 9 males) were painted with fraction
“ C ” as in Group I. After an interval of 3 weeks they were painted with croton
oil t-wice weelclj' until death.

Group IV.—Tu-enty-two mice (8 females, 14 males) Avere painted 3 times a
AA-eek, for 2 Aveeks only, Avith neutral fraction. They received no further treatment.

Group V. — TAA^enty-one mice (6 females, 15 males) Avere painted Avith neutral
fraction as in Group IV. After an interA^al of 3 Av^eeks they Avere painted AAith
fraction “ C ” 3 times a AA'eek until death.

Group AT. — Twenty -five mice (1 1 females, 14 males) Avere painted iw'th neutral
fraction as in Group IA^ After an interval of 3 Aveeks, they were painted with
croton oil tAA'ice Aveekly until death.

Group ATI. — TAA'enty-three mice (13 females, 10 males) were painted with
croton oil tAA'ice Aveeklj'' until death.

RESULTS

The results are summarised in Table I.

Group /. — The dose and duration of painting AA'ith fraction “ C ” Avere arbit-
rarjL It Avas knoAvn to produce many skin tumours in C57BL mice AA'hen applied
throughout life (Clemo and Miller, 1957). From the present experiment it was
evident that a minimal carcinogenic dose had been given. Taa'o males (aged 1ft
and 29-5 months) developed 3 small papillomata, 2 of AA'hich, at the site of painting,
Avere superficial, Avhile in the third (slightly larger and on the abdominal surface)
the epithelium had not yet reached the panniculus carnosus. One female deA'e-
loped a rapidly groAA'ing spindle-celled subcutaneous sarcoma over the left scapula,
at the age of 20 months. The non-tumour mice all lived well into tumour age,
4 djdng Avith leukaemia, 2 AA'ith hepatomata and one with leukaemia plus hepa-
toma.

Group II. — Three males dcA'eloped cpitheliomata (one per mouse) at the site
of painting, all groAA'ing steadily so that the mice had to be killed 3 to 4 months
after the first appearance of the lesions ; 7 papillomata appeared in 5 other males,
all at the site of painting. In 4 females there Avere 8 skin tumours (one mouse had
4 and in another mouse 2 coalesced to form one) ; of these, 5 were innocent
papillomata but 3 AA'ere classified as “ probablj' malignant Non-tumour mice
liA'ed Avell into tumour age ; one female died at 1 1 montlis of leukaemia, the other
at 15-5 months of pneumonia, AA'liile gross kidney disease caused the deaths o
the 7 non-tumour males.

Group III. — The maximum total numher of skin tumours in the 9 tumour
females Avas 14, but 4 disappeared before death, leaA'ing a final total of ^ ®
greatest indiA'idual number being 4 ; of these 1 0 , one grew slowly but steadi j
to become “ probably malignant ” at the time of death 8 months later, the res
remaining small. Tlie three tumours in males (one each) AA'ere all small, f w
non-tumour mice Avere all of tumour age and the majority died with gross}

diseased kidneys. . -i.

Group IV. — In this group 2 male mice each developed one small skin P^P ,
loma ; one of these, in the lumbar region, disappeared before death ;

AA'as in the centre of the abdominal surface. The remaining mice died a
age AAuthout skin tumours but 3 had leukaemia, one a A^ery large lung ™ ^
(rare in this strain), one had haemangioma of the spleen, one a hepatoma an
rest had diseased kidneys.

654

G. E. CLEMO AND E. W. MILLER

Group ^ . The majority of the mice, otherwise healthy, were killed when their
tumours became large and malignant or “probably malignant”, but two had
kjdney disease and died before their tumours had grown to any size. Every
mouse had multiple skin tumours. The tumour incidence in males and females
]s given in Table II. Although the males had on an average more tumours per
mouse than the females, the difference was not significant either for the maximum
average numbers (column 5) (d = 1-3, 2 x S.E. = 2-1) or for the final average
numbers (column 6) after tumours had coalesced (d = 1-5, 2 x S.E. = 1-9).
But when the proportions of tumours which became malignant or “ probably
malignant ” n^ere compared (column 11), the difference between the sexes was
significant (d = 36-5, 2 x S.E. = 28-6). The average age of the females at
tumour appearance Avas 10-8 months (range = 9-0-12-5 months) and that of
the males was 9-8 months (7'5-ll-5 months) ; the average age of the females at
death was 14-2 months (12'0— 17-0 months) and of the males was 14'5 months
(11'5-17'5). Thus although the latent period was longer in the females, the
tumours became malignant more rapidly than in the males, and a greater propor-
tion of the tumours became malignant (and “ probably malignant ”) in the
females.

Group FI. — TJie only mice to produce skin tumours were 2 males, each having
one small papilloma, one at the age of 14 months (on the forehead) and the other
at 17 months (interscapular) ; the latter had disappeared at the time of death 2
months later. The mice in this group, although of tumour age, died sooner than
those of other groups, most of them with diseased kidneys.

Group VI I . — There was only one tumour mouse, a female with a skin papil-
loma on the left flanlc at the age of 21 months. That tumour disappeared before
death 7 weeks later, but meanwlule another papilloma, which had appeared in the
dorsal region a week or two after the first, had developed into a small epithelioma.
The remaining mice died tumour-free at a similar age to those in Group VI and
all but one had grossly diseased kidneys.

DISCUSSION

It is clear from these experiments that, under the conditions of treatment
described, the neutral fraction of cigarette smoke can act as a definite tumour
promoter. Some evidence has already appeared in the literature that whole
tar from cigarette smoke may have tumour-promoting properties when applied
after a knoum carcinogen such as 3 : 4-benzopyTene (Hamer and Woodhouse,
1956 ; Gu’ynn and Salaman, 1956) ; these workers found no proof of tumour-
initiation. Gellhorn (1958) demonstrated convincingly the tumour-promoting
activity of whole tobacco tar after the preliminary application of 3 : 4-benzo-
P3n’ene ; he found also that although croton oil promoted a higher incidence o
carcinomata, tobacco tar increased the “ conversion rate ” of carcinomata from
papillomata compared with croton oil. More recently Roe, Salaman and tolien
(1959) have shown that the phenolic fraction is a tumour promoter, the initiator
in this case being 9,10-dimethyl- 1,2-benzanthracene ; they proved also that the
neutral fraction is carcinogenic. Wynder and Hoffman (19 ^ ■

tumour-promoting properties of the phenol fraction and of the mco me re

A^fi as is known, the present series of experiments is the first in *5®

initiating substance has been, not one of the weU-known carcinogens such <-s

TUMOUll promotion UV CICJARRIIR SMOKli

055

“ C ” uionc and any LypotLclical pro, not ing
effect due to the prehminarj^ treatment with ncntral fractum would he <pnl<!
obscured by the potency of fraction “ C That, a minnnal initial mg dose was
given to the mice in Group I was shown by the very low tiiinonr inculeiiee m that.

^ "nie crucial results are given by Groups II and 111 ; in these, fraction (■
applied in minimal dose as initiator was followed in Group II by neutral fract ion
and in Group III by croton oil as promoters. Croton oil is a well-known t ninonr
promoter ; it has also been showm to be a weak carcinogen, producing a nninber
of papillomata which usually regress when treatment ceases, but. lloe (10.1(5)
reported 7 malignant tumours in 20 mice which had been treated for from to^
72 weeks. In the present instance, applied alone throughout life (nji to 21 inoiit bs
treatment), it produced in Group VII only one malignant tumour and one jmiiil-
loma which regressed in 23 mice living over one year.

In Group III 12 of the 24 mice developed a total of 10 skin tmnour.s, of which
4 regressed and only one (which took 6 months to grow and was the only t iiiiioiir
of any size in that group) became malignant ; the final tninonr incidence was 33
per cent.

In Group II 12 of the 21 mice produced a total of 18 skin tumours, of which
none regressed (but 2 coalesced as they increased in size) and (5 became malignaiit
or probably malignant, 5 of them attaining quite a large size in from 2 to 4 months.
Thus although the latent period (see Table I) was much longer with neutral
fraction as promoter, there was a much shorter time between tumour appearance
and death than with croton oil as promoter. While the differences in tumour
incidence are not statistically significant, a comparison of the charts of tiimoiir
growth was most convincing and showed that under the conditions of the e.vperi-
ment a 10 per cent solution of neutral fraction applied three times a week was a
more powerful tumour promoter than a 0-5 per cent solution of croton oil applied
twice weekly.

The present experiments provide less certain evidence of tumour intiation by
neutral fraction ; noth what was intended to be a minimal dose (Group IV)
papillomata appeared in 2 mice out of 22, but one regressed and the other was far
wl\ ^fter this preliminaiy treatment

o^v Tn I "'I' ° ^™"P promoter, again

onlj 2 mice de% eloped skin papillomata, no more than might be expected from the

use of croton oil alone. While this might mean that the originS d^e too
■small to gme umours, in previous experiments in which a 10 per Tolution
of neutral fraction was applied tlrroughout life to mice of the A ami G57PT •
no skin tumours were obsen^ed. On the other Wl Pne ^

(fro,.. „ .t 30 being

applied neutral fraction thre; fdoS

40 mg. at each painting.

656

G. R. CLEMO AND E. W. MILLER

In the present work the dose of neutral fraction, forming about 18 per cent by
weight of the Avhole original tar (Clemo, 1958), was approximately 6-I ms per
mouse at each painting. The neutral fraction used by Roe, Salaman and Colien
(1959) which apparently contained the ester fraction removed from our neutral
fraction, formed approximately 56 per cent by weight of the original tar. It is
thus not possible to compare strictly the individual doses used by ourselves and
by Roe e( al. (1959). Gellhorn (1958) gave approximately 50 to 60 mg. of tar
per week to each mouse ; our dose of 19-2 mg. neutral fraction per week probably
represents about twice as much neutral fraction as would be present in his dose.
Althougli Gellhorn used the same strength of croton oil, he was giving three times
as much per week as in our experiments ; he obtained far more tumours with
croton oil than he did with the tar (as promoters), and given the differences in
dosage the present results are not inconsistent with his.

It was noticed in the present experiments that mice receiving croton oil
throughout their lives (Groups III, VI and VII) died much earlier than those in
other groups (except Group V where they all developed tumours) and the majority
had severe kidney disease.

SUMMARY

Experiments are described in which a definite tumour-promoting effect was
observed when the neutral fraction from cigarette smoke was applied to C57BL
mice after an initiating dose of fraction “ C ” from city smoke. Eighteen skin
tumours, of which 6 became malignant or “ probably malignant ” were produced
in 12 out of 21 mice painted in the interscapular region throughout their lives
(up to 28 months). Similar treatment with croton oil after fraction “ C ” resulted
in 16 skin tumours, of which 4 regressed and one became malignant, in 12 out of
24 mice.

There was little evidence of either a tumour-initiating or a complete carcino-
genic effect with a small dose of neutral fraction. No more tumours were pro-
duced with croton oil applied after neutral fraction than were produced by croton
oil alone.

When fraction “ C ” was applied throughout life, the latent period was longer
in females than in males, but tumour growth was more rapid in the females and a
significantly greater percentage of tumours in females developed to malignancy.

This work was carried out •vvith the aid of a research grant from the North
of England Council of the British Empire Cancer Campaign, for which the authors
would express their gratitude. Thanks are due also to Mrs. Eileen Moody of the
technical staff for her assistance.

REFERENCES

Clemo, G. R. — (1958) Tetrahedron, 3,168.

Idem AND Miller, E. W. — (1957) Brit. J. Cancer, 11, 403.

Jidem and Pvbus, P. C. — (1955) Ibid., 9, 137.

Gellhorn, A. — (1958) Cancer Res., 18, 510. „ . „ „ lOl

Gwynn, R. H. and Salaman, M. H.— (1956) Rep. Brit. Emp. Cancer Campgn., 34, IJ3.
Hamer, D. and Woodhouse, D. L. — (1956) Bril. J. Cancer, 10, 193.

Kotin, P.~-(1956) Cancer Res., 16, 375.

Roe, F. j. C.— (1956) Bril. J. Cancer, 10, 72.

Idem, Salaman, M. H. and Cohen, J.— (1959) Ibid 13, 6M.

Wynder, E. L. and Hoffman, D.-{1960) Proc. Amer. Cancer Res., 3, 104.

the of

J. S. HOWELL

From the Deparlment of PnUiologu and Cancer ItcMarch, The Medical Selmd.

Birmingham

Received for jiuiilieotion Sepletniter 23.

limo

5 *Ia 3 CvIary tumours in the rat can he induced hy a variety of - ^

include intensive treatment with liormonc preparations c.g. K’''"'' *’ J’”" '
(Moon et al, 1950) and oestrogens (Geschickter, lOSt) ; Mackenr.o > 00 ). l.v the
administration of aminofluorene compounds (Biclschowsky. HIU, l-M < : ^yI^(•o-
nidis, 1954) and by certain carcinogenic hydrocarbon.s. Thc.se latter snlistancc.s
have been administered by various routes, but the most rapid met bod of uuniot urn
has been reported by Huggins, BriziarelH and Sutton {10510 giving incthyleholan-
threne daily by stomach tube, a technique originally described hy Shay r/ r;/. ( 1 !)1!)).

Painting the skin of the rat with an oily solution of 9 : U)-dinicthyl-l : 2-
benzanthracene (DIilB) at fortnightly intervals is a highly effective method of
inducing breast tumours. In initial experiments (Howell, 1959) it was shown that.
77 per cent of female rats developed mammary tumours in an average time of
4-75 months, and furthermore, a single application of the carcinogen gave a tumour
incidence of 75 per cent, although in this instance the average induction time was
extended to 12 months. The present paper is concerned with the efrccts of gouad-
ectomy, hormone supplements and the effects of normal and pathological lactat ion
on tumour development following skin application of DMB. The experiments on
lactation were undertaken since Marchant (1958) has shown that breast tumour
development in IE mice is inhibited by full lactation, and that unilateral removal
of the nipples in lactating IE mice allows the development of breast tumours on
the side without nipples, but not on the normal side (Marchant, 1959).

3IATERIALS AND METHODS

General

The animals were derived from two sources ; entirely outbred laboratory
stock, and from the Birmingham strain (Laboratory Animals Bureau Catalogue of
Uniform Strains, Ho. 626, 1953). ^

f tile animals whose age at the start of carcinogenic treatment

^ months were kept in galvanized wire-mesh cages never
more than o rats to a cage, and were given rat cube (Heygate & Sons knfwn as tlie
Thompson diet) and water ad lib. Twenty drops of a l-G per cent SuZ nf UM?

m oh\ e oil was applied to the skin at fortnightly intervals 5 drons tn K •

the ventral and dorsal surfaces • n slnnriB ® to each side of

i r ’ ^ Single treatment averaged 1*3 ml or tykt

658

J. S. HOWELL

although sometimes the tumours were allowed to grow. In animals not developinff
breast tumours, treatment was continued until death, or until excoriation of the
skin, sometimes with tlie development of skin tumours necessitated stoppine
trcci t jn G17 1/ .

Experimental groups

Experiment In this experiment a total of 35 laboratory stock males and
38 Birmingham strain female rats divided into 5 groups were used ; the appropriate
treatment of each group is detailed in Table I. Castration or ovariectomy was

Table I (Experiment A). — Experimental Groups and Results

Number Rats
of “ at

Rats risk ”

Group Treatment F 31 F 31

1 . D3rB only , . . 22» 8 . 22 8

2 . Gonadectomy + D3[B . 9 9. 99

3 . Hexoestrol -f MIB .10 9. 75

4 . Proge-sterone + D3IB . 10 — . 6 —

u . Hexoestrol +

Progesterone + D3IB . 9 9. 99

Average

Average time

induction Average to

Breast time number death

tumours (months) paintings (months)

F 31 F 31 F 31 F 31
17 0 . 4-75 — . 11 18 . 5-3 9-3

0 0 . — — . 16 17 . 8 8-8

2 0 . 7-2 ~ . 14 15 . 7-2 7-5

3 — . 7-8 — . 18 — . 9 —

51.7 9 . 14 15 . 7 7-5

* = Animals of previous experiments
Hexoestrol = 7 mg. pellets

Progesterone = 5 mg. intra-peritoneal injection everr* 14 days.
All groups received D3tB every 14 days.

performed when the animals were 6 weeks old, and treatment unth the carcinogen
was commenced 4 weeks after the operation. The hexoestrol pellets, 7 mg. each
(Boots Pure Drug Co.), were inserted at 4 weeks of age by trochar and cannula
into the subcutaneous tissue between the scapulae ; D3IB painting was started
4 w^eeks following the implant. The animals in these groups surviving 4 months
or more received a second 7 mg. implant. Progesterone, given to some animals,
was administered by intra-peritoneal injection, in a dose of 5 mg. dissolved in
olive oil at fortnightlj’^ intervals, alternating with the application of DMB.

Group
6 .
7 .

Table II {Experiment B). — Experimental Groups and Results

Number

Treatment Rats

Lactation + DMB . 12

Lactation -f Uni-
lateral nipple
removal I13IB . 12

Average Average Average

Number induction number number

“ at Breast time of of

risk tumours (months) litters paintings

11 . 1 . 6 . 3 . 17

10 . 3.8-8. 3

Average

time

death

(months)

8-8

7-0

Experiment B. — ^In this experiment (Table II) 24 laboratory stock e
were used ; they were divided into two equal groups, and the animals wer p
in individual cages. One group of 12 rats were mated, and a ter ^c i j

and suckled a litter, treatment with the carcinogen was started. Ike

IKDUCTION OF HAT UllKAST TOMOrUS

or.n

p„„p,vas treated identicallpscort*^^^^^ aide

were excised following suckling Throuchoul. liotli these exiierinients. hreeding

could not be suckled thereafter Th ^ carcinogen trealnienl was

SS"d teratCeati: or mdil the a, ale of , lie aid,, I

lulling them.

Pn*?/ 7iioYl£.iifi ci7id /n*s/o[o(jficn? methods i r •

ajS£^“=£53S=-

tissue was also preserved from all organs showing gross lint hologienl ( hang< s.

All tissues were fixed in 4 per cent formnldchydc-sahne. Sections were
with Ehrlich’s haematoxylin and eosin, Wcigert’s hncnintoxyhn and nn . so
and by Lawson’s elastic stain. On occasion frozen sections were cut and stained

for fat.

besults

The results of Experiments A and B arc detailed in Tables 1 and 11.

Experiment A {Table I)

Group I . — ^Eight males survived for an average period of Hot months, receiving
an average of 18 paintings each. None developed breast tumours although skin
tumours were present in 7. One had a squamous cell carcinoma of the car duct.

The results for normal females have been abstracted from previous cxjieriincnt s
already reported (Howell, 1959). Suffice it to state here that 22 femnles were
treated noth the carcinogen and 17 developed breast tumours in an average time
of 4-75 months.

Group 2. — ^The 9 castrated males survived for an average period of 8-8 months,
receiving an average of 17 paintings ; 5 developed skin tumours and 2 develojicd
squamous carcinomata arising from ear ducts, but no breast tumours were found.

Nine ovariectomized females survived for an average period of 8 months,
receiving an average of 16 paintings but despite this no animal developed a
breast tumour. Seven developed skin tumours and 4 had squamous carcinomata
arising from ear ducts.

Group 3. Survival of the male animals in this group was poor ; 4 died after
only 2 or 3 paintings. The remaining 5 survived for an average period of 7-.5
months, receiving an average of 15 paintings, hut none of them developed breast
tumours. Two had skin tumours. ^

in the females was better, but 3 animals were lost due to cannibalism
after treatment had been m progress for 6 months, none of which had had nalnible
breast tumours before death. The remaining 7 were treated fnr - 9 ^ !

found at 9 months. None had skin tumours. ^ ® P’

6 I,™ treated tor an arerage period ot 9 months recoirint In’

660

J. S. HOWELL

paintings. Two developed breast tumours at 6-25 months and a further animal
deveioped a breast tumour after H months, tlus animal was the sole sunnvor
None had sldn tumours.

Group 5.— Nine males survived for an average period of 7-5 months receiving
an average of 15 paintings. One animal developed a breast tumour at 9 months.
Two other animals had skin tumours, and 2 had squamous cell carcinoma of the
ear ducts.

Nine females survived for 7 months and of these, 5 developed breast tumours,
the first appearing after 3-5 months and the last one after 10-5 months. The average
tumour induction time was 7 months. Two rats had skin tumours, and one had
a squamous cell carcinoma of the ear duct.

Experiment B {Table 11)

Group 6. — Eleven rats survived for an aAmrage period of 8-8 months, during
udiich time they received an average of 17 paintings and produced and suckled an
average of 3 litters per rat ; one rat had 5 litters, 5 had 4 litters, one had 3 litters,
one had 2 litters and 3 had a single litter. One rat died early in the experiment
due to sepsis in the genital tract. One breast tumour was found in the rat that
had had 2 litters ; this was of an unusual appearance on gross examination and
was subsequently slio^vn to be a simple fibro-adenoma. Seven rats developed skin
tumours and 4 had squamous carcinomata of the ear ducts.

Group 7. — Ten rats survived for an average period of 7-5 months. In this time
they received an average of 15 paintings and produced and suckled an average of
3 litters per rat ; two rats had 5 litters, one had 4 litters, 4 had 3 litters, 2 had 2

EXPLANATION OF PLATES
Fig. 1. — Nomal female rat breast. H. and E. x37.

Fig. 2. — ^Duct in normal female rat breast showing elastic fibres in the wall. Elastic X 73.
Fig. 3. — ^Normal male rat breast shoiring solid acini. H. and E. Xl8.

Fig. 4. — Dilatation of ducts filled with globules of secretion. Female -n-ith hexoestrol implant.
H. and E. X 75.

Fig. 5. — Lnctating DJIB-treated breast. H. and E. X73.

Fig. 6. — ^Lactating DMB-treated breast. To show cystic dilatation of a duct on the side from
which the nipples had been removed. H. andVG. X73.

Fig. 7. — Poorly differentiated carcinoma of breast. H. and E. X 75.

Fig. 8. — ^Duct-like structures in carcinoma of breast. H. and E. X 87.

Fig. 9. — ^Intracystic papilliferous gron-th. H. and E. x87. tt a

Fig. 10. — Trabecular groivth showing resemblance to human scirrhous carcinoma. H. and E.

Fig. II. — From an area of alveolar-like growth showing secretory changes in cells. H. and E.
Xl87.

Fig. 12. — ^Invasion of muscle. H. and VG. x87.

Fig. 13. — ^Fragmentation of elastic fibres round ducts. Elastic x8 (. ^

Fig. 14. — Fragmentation of elastic fibres around a duet. The elastic tissue appears as djstmc
blobs in the wall of the duct. Elastic X335.

Fig. 15. — ^Early proliferation of cells lining a ductule. H. and E. Xl8<.

Fig. 16. — Marked cellular proliferation within a ductule. H. and I G. X I <6.

Fig. 17. — ^Intraduct carcinoma. H. and E. X75. nt

Fig. 18.— Intraduct carcinoma with early infiltration. Note disruption of elastic fibres
point of infiltration. Elastic XllS.

Fig. 19. — Squamous cell carcinoma of ear duet. H. andE. X95. vr vlS

Fig. 20. — ^Fibrosarcoma. Note dermal appendages included m the tumour. H. and v
Fig. 21.— Hair follicles within a fibrosarcoma. H. and VG. x 140. jj andE

Fig. 22.— Cystic dilatation of Zymbal’s gland associated with squamous metaplasia. H. and E.

X32.

BltlTISIl JorUXAI. OK Cam k.i!.

Vol. NIV. -''O-

II.

15i:itisii .loi t.nai. or

Vol. S«. I.

Howoll

Ofil

IICDUCTIOX or RAT BREAST TUMOURS

litters and one liad a single litter. Two rats had died early as a rcs,dt of scij^sis in
the genital tract. Three animals developed breast tnmonrs ; one of found

at 11 months, was on the side with intact nipj)lcs in a rat that had had o litters.
The other 2 animals developed breast tumours at 7-75 months on the side from
which the nipples had been removed. One of these rats had .$ separate breast
tumours and had had 3 litters ; the other rat with a single breast t umour lifRl hi»l
4 litters. Two rats had shin tumours, and one had a stpmmous carcinoma ot the
ear duct.

Palhology

Normal female and male rat breast

The adult virgin female rat breast has well developed ducts within the lumen
of wliich is frequently rather dense eosinophilic secretion. The ducts are lined
internal!}’ b}' a single layer of cuboidal cells, external to which is a layer of myo-
epithelium surrounded by a fibrous mantle which decreases in thickness as the
ducts branch and become smaller (Fig. 1). Elastic fibres are present within the
fibrous tissue encircling the ducts, and by definition a duct must have elastic
fibres in its wall (Fig. 2). Arising from the ducts arc small bud-like projections
forming acini frequently with ill-defined lumina. The acinar cells, and frequently
the cells lining the ducts, may contain secretory vacuoles.

The male rat breast compared with other male rodents shows an unusual
degree of development throughout adult life. There are fewer ducts as compared
with the female, but solid acini are more numerous, the acinar cells being large
and eosinophilic (Fig. 3). Both ducts and acini contain eosinophilic secretion, but
no evidence of secretorj’- activity was observed in the lining cells.

Histology of breasts of DMB-treated animals without tumours

Ovariectomy made comparatively little difference to the histological structure
of the breast except that duct and acinar development was slightly less than in the
intact rat. The cells forming the acini wmre larger and the endoplasm was more
eosinophilic than in the intact rat and they showed no evidence of secretorj’
activity. Castration was also without marked effect on the histological structure
of the breast except that in some animals acinar development was less than in
the intact male.

Females with hexoestrol implants showed great proliferation and extension of
e duct systems, w-luch were also greatly dilated and filled with multiple globules
0 dense, eosinophilic, sometimes basophilic secretion (Fig. 4). The cells lining
showed marked secretory activity. The fibrous stroma of the
increased in amount, replacing the normal adipose tissue and the fibrous

cells wem°nrp^ acellular. Chronic inflammatoiy^

acini hut Initially there was also some proliferation of

tissues overshadowed by the duct development. The breast

tSroflhTtm^r^^^ - appearance to

* progesterone injections gave the

procestprnrrr,^ picturc in both females and males as with hexoestrnl oinroo
crone .pp„e„tly nrade no difference to breast UstoC^te feSS

662

J. S. HOWELL

receiving progesterone injections without hexoestrol showed similar histolomcal
appearances to the females without hormonal supplement. ®

I females that were allowed to breed and lactate during carcinogen treatment
showed changes m the breasts dependent upon the stage of pregnancy or lactation
at which they were killed or died and no abnormalities of the breast tissues were
observed (Fig. 5).

In the rats that had had all the nipples removed from one side of the body, the
contralateral breast showed normal pregnancy or lactational changes. On' the
side with the nipples removed, the breast tissues showed changes of pregnancy or
lactation but these were not so well developed as those on the normal side and in
some of these animals there was very considerable cystic dilatation of ducts which
were filled with globules of dense secretion (Fig. 6).

Breast tumours

The breast tumours were first identified as small, discrete, freely mobile nodules
in the subcutaneous tissues. They tended to grow fairly rapidly and, if the animal
was not killed, they attained a considerable size. They could usually be dissected
from the surrounding tissues with ease ; fixation to the deeper tissues was
uncommon, but fixation to skin, sometimes associated with ulceration occurred
frequently, especially in the larger tumours.

Oji gross inspection, the tumours had a smooth, sometimes lobulated surface.
Thej' were usually yellowish-pink in colour wth a soft fleshy consistency, and the
reshly cut surface frequently exuded a small quantity of a pale milky fluid.
In the larger tumours areas of cystic degeneration rvith and without haemorrhagic
necrosis were common, sometimes Avith superimposed infective changes.

On histological examination, the breast tumours with a feAV exceptions were
adenocarcinomata, Avith a complex histological structure Avhich varied not only
betAveen tumours but also between different parts of the same tumour. ,

The tumours showed all degrees of differentiation ; frequently they consisted
of solid sheets of cells AAuth scanty stroma and no eAudence of differentiation
(Fig. 7). Usually lioweAmr they shoAved closely applied duct-like structures with
a cylindromatous appearance, the intimate structure of Avhich had a solid or
cribriform pattern (Fig. 8). Sometimes cysts were present associated Avith intra-
cystic papillary groAvth (Fig. 9). Not infrequently areas of solid acinar and trabe-
cular groAvth AAmre present ; these Avmre usually situated at the periphery of the
tumours and the appearances Avere somcAA'hat similar to the human scirrhous breast
carcinoma (Fig. 10). The stroma in these tumours Avas fairly abundant, consisting
of cellular fibrous strands closely investing the epithelial elements. Sometimes,
thicker and more hyaline fibrous trabeculae transected the tumour giAung nse to a

distinetly lobular appearance. i ■ i

In other types of tumour, ahmolar-like growth Avas observed in Avlucn tne
intimate structure showed a resemblance to the lactating breast, with secre ory
vacuoles in the epithelial cells lining the alveoli (Fig. 11). The stroma m t js ype
of growth was scanty and consisted of thin fibrous strands separating alveo i rom
each other, although sometimes coarser bands Avere also present breaking tne

Unlike the chemically induced mouse breast tumours squamous
rare ; when present it Avas always adjacent to an area of necrosis or o er in <

IKDUCTION OF RAT BREAST TUMOURS

(503

„.atory motion. Metastaseo in Ijnnpl. glands or oWm organs rvero never observed

ftagmentotion and condensation of tlic elastic fibres around the ducts, so that tbo
Stic tissue appeared as distinct blobs (Pig. 13 and 14). In some ducts there ua.s
proliferation oFtbe lining cells, so that these became 2-3 cells thick and alniost
filled the lumen (Pig. 15 and 16), in certain instances this change progrc.sscd tn
frank intradnet carcinoma sometimes associated with invasion of the duct nail
and early infiltration of the stroma (Pig. 17 and 18). Another change consisted of
the development of acini which frequently had vacuoles m the lining cells ; thc.so
appeared to bud out from ducts and appeared more marked than acinar develop-
ment in the normal virgin rat breast.

Other pathological changes

In addition to breast tumours, certain other neoplastic lesions were found in
these rats. The commonest, found in rats of most groups, were skin tuniours,
which histologically were mainly kerato-acanthomata. Tliej' tended to develop
later than the breast tumours and were most numerous in animals without breast
tumours. In 15 rats, 5 male and 10 female, squamous cell carcinomata arising in
the ear ducts were found (Pig. 19). These produced large swellings adjacent to the
ears and at post-mortem examination were nearly always secondarily infected.
In one of these animals there were massive deposits of secondary squamous cell
carcinoma in both lungs, which at first, on gross inspection, was confused with
bronchiectasis, but histology showed undoubted carcinoma.

In 12 rats there were lymphomatous lesions, characterized by pallor and en-
largement of lymph nodes, thymus, liver and spleen. Histologjf showed destruction
of the normal architecture of these organs rvith replacement and infiltration by
primitive cells, the appearances of which were suggestive of white cell precursors.

In 6 rats, 4 males and 2 females, interesting fibrosarcoraatous tumours were
found (Fig 20), consisting of interlacing whorling bundles of collagen whicli varied
considerably in cellularity. Some were hyaline and relatively acellular but others

fibrohlast-like cells showing occasional
mitotic figures. Ilithin the tumours there were sometimes occasional epithelial
remnants suggestive of breast ducts which appeared to be incorporated by hifiltri-
tive and expansive groivth of the tumour. Since hair follicles and Slier ski, i
appendages were sometimes also incomorated CPio- 9 ii fVio’ ^ ^ ^

immediately subjacent to the epidermis. ^ PPearea to arise m the dermis

enlarged pituitary gL^J^^lSfobgicdIy*t£eSf^^^^^ hexoestrol implants had

, discussion

reported e,fpTJU”„ “TtJTe Multta rf «

Dreast tumours since the

664

J. S. HOWELL

carcinogen and/or its mode of administration differ, as do the various hormone
preparations used.

Shay, Harris and Gruenstein (1952) and Shay, Gruenstein and Harris (1956)
administered 2 mg. of methylchoJanthrene daily by stomach tube to normal
female rats and 89 per cent developed breast tumours in an average time of 6-5
months. The age of the animal at the start of treatment had some bearing on
tumour yield and induction time, since using older animals, the tumour incidence
fell, and the average induction time increased. Employing the same technique
but with rats of 42 days old and increasing the daily dose of methylcholanthrene
to 10 mg., Huggins, Hriziarelli and Sutton ( 1959 ) obtained breast tumours in
every animal treated in a mean time of 55-9 days. This represents one of the
most rapid methods of obtaining the tumours. These results can be compared with
those obtained by applying HME to the skin ; with a 0-5 per cent solution of the
carcinogen, 75 per cent developed breast tumours in an average time of 7-25
months, wdiereas using a T6 per cent solution, the average induction time fell
to 4-75 months although the tumour incidence was unaltered (Howell, 1959).
Geyer el al. (1953) using intravenous DMB also showed that the quantity of
carcinogen administered influenced the jneld and induction time of tumours.

In the present experiments DMB-treated normal male rats did not develop
breast tumours. Geyer el al. (1951) also failed to obtain breast tumours in males
following intravenous DMB, as did Dao and Sunderland (1959), and Kim and Eurth
(1960) giving intragastric methylcholanthrene. Shay el al. (1952) found that 43
per cent of normal males developed breast tumours in 12'8 months, but only one
was a tj^ical adenocareinoma, the remainder being spindle cell or collagenous
tumours, and in one instance a fibro-adenoma. In 3 intact males in the present
experiments fibrosarcomatous tumours were found, but these are not considered
to be breast tumours within the context of this paper.

Ovariectomy follou^ed bj'^ DMB completely inhibited breast tumour develop-
ment despite continuation of treatment for an average period of 8 months. Shay
el al. (1952) obtained breast tumours in 37 per cent of ovariectomized females,
but the average induction time w'as 12-8 months, rising to 15-5 months in older
animals. Huggins, Briziarelli and Sutton (1959) also observed a reduction in
tumour incidence folloiving ovarieotomj'.

No breast tumours Avere produced in males that were castrated then given
DMB. No details have been found in the literature concerning the effect of castra-
tion on breast tumour production following administration of carcinogenic hydro-
carbons except for Dao and Sunderland (1959) who obtained tumours in 2 castrated

animals. . ,

When intact females were treated with hexoestrol implants concurrently witli
the carcinogen only 2 out of 7 rats developed tumours. Although these observa-
tions are based on a small number of animals they tend to support Shay el a.
(1956) who found that oestrogen caused a sharp fall in tumour incidence and
prolongation of average induction time and they made the interesting suggestion
that the fall in tumour incidence might be due to development of oestrogen induced
pituitary tumours with consequent “ physiological h 3 ^ophysectomy ,
rendering the animals insensitive to the carcinogen. In this connection i is ®’§n!
Scant that hypophysectomy completely inhibited °

following intramuscular injections of DMB (Noble and Balter^ o )
follondng intragastric methjdcholanthrene (Huggins, Grand and Bn an ,

INDUCTION OF RAT BREAST TUMOURS

0G5

Intact DM-trcated males with 'ner*trof‘'oc*rog^^^^^^^^

-“tt" retcn«otf 8V'7 or ntalc

Progesterone injections plus MIB were given to female rats only, and .1 out of 0
animals® developed tumours in 7-8 months, however the do.se of I>™eof7>’o ' ' 1
was substantially less than in other experiments reported m tlie litciatmc blinj
et al. (1952) with a 50 mg. progesterone implant observed a moderate fall in tumoi r
incidence with a slightly prolonged average induction time. However, Huggins,
Grand and Brillantes (1959) giving 4 mg. of progesterone daily, obtained breast
tumours in all the animals treated wdth a reduction in average induction time u Inch
was even greater using the synthetic preparation 9-2 Bronio-ll-ketoprogcstcronc.

The combination of hexoestrol, progesterone and DMB gave breast tumour.s
in 5 out of 9 female rats after an average induction time of 7 montlis. Unlike the
groups given the hormones separately, the incidence of breast tumours ajiproached
that observed in rats treated with DMB alone, although the induction time was
2-25 months longer. Scholler and Carnes (1958) found no effect on tumour pro-
duction when intravenous DMB was given with oestrogen and progesterone.

Hine male rats were treated for an average period of 7'5 months with the com-
bined hormones and DMB ; one animal, the sole survivor, developed a breast
tumour after 9 months. This suggests that by suitable hormonal preparation
breast tumours can be induced in the male. In this respect it has been shown t hat
the relative proportion of oestrogen to progesterone is of crucial importance in
the development of the normal breast (Folley, 1952) and hence the proportions of
the hormones given to animals in carcinogenesis experiments may be of similar
significance.

It is commonly accepted that the incidence of carcinoma of the breast in the
human female is reduced by breast feeding, and experimentalljf, Marchant (1958)
has shown that breeding with lactation reduces the incidence of ehemically
indued breast tumours in IF mice. In the present experiments, breeding, lactatioii,
and miB treatment were continued for an average of 8-8 months but only one of
11 rats developed a tumour, found at 6 months ; this was a fibro-adenoma in
contrast to the adenocarcinoma usually induced. Dao and Greiner (1960) iisiim
intragastric methylcholanthrene also observed a marked fall in tumour incidencf
when rats were allowed to breed and suckle their young. mciaence

Breast tumour development following skin application of DMB is nrnbabiv
ment in lactating animals mav bp that th ■ breast tumour develoj)-

leaving insufficient time for it to L on thfbrrrr^^'' is excreted in the milk

shown that methylcholanthrene is excreted in the mfik f
ments milk expressed from lactating breasts M DMrV 'I
in ultraviolet light. ^ ® DMB-treated animals fluoresced

666

J. S. HOWELL

status and milk excretion, a technique described Marchant (1959) using IF
mice. The results however are inconclusir^e since only 3 animals developed tumours.
Two tumours, found at 7-25 months, were on the side from winch the nipples had
been excised ; the third rat had a tumour on the normal side but this did not
develop until 10 months. It may also be significant that one of the animals with
tumours on the side without nipples developed 3 distinct tumours, all foimd at the
same time.

The ear duct tumours observed in 15 animals were aU squamous cell carcino-
mata apparently arising in Zymbal’s gland, a sebaceous gland situated near the
tympanic membrane. Cj'^stic dilatation of the gland associated with squamous
metaplasia of the lining epithelium (Fig. 22) preceded tumour development.
>Similar tumours have been produced by 2-acet3daminofluorene (Bielschowsky
1944, and Skor5Tia, Ross and Rudis, 1951) and intranasal instillation of DMB
(Howell, unpublished observations). Since the breast is a modified sebaceous
gland, it is perliaps not surprising that tumours should also arise in Z^unbal’s
gland, although these tumours have not been reported following intragastric
methjdcholanthrene.

STJMJIAKy

1. Experiments are described which show that painting the skin of female rats
with an oily solution of Dj\IB is an effective method of inducing breast tumours.
It has been shoum that hormonal factors are important for tumour development ;
ovariectomjq continued breeding and lactation prevent their development and there
is a suggestion, based on a small number of animals, that hexoestrol implants
with and without progesterone injections I'educe the tumour incidence and prolong
the induction time.

2. Experiments have shown that male rats do not develop breast tumours,
neither do castrated males. Hexoestrol implants were m’thout effect on breast
tumour development, but one out of nine male rats given both hexoestrol and
progesterone developed a single breast tumour.

3. Certain other neoplastic changes in rats treated uith DMB are briefly
described.

4. The results of these experiments are compared with other results on the
induction of rat breast tumours reported in the literature.

I am grateful to Professor J. W. Orr for helpful advice and criticism, I am
also grateful to Dr. A. D. Hudson for help in the early stages of tliis investigation.

thanks are due to the Birmingham Branch of the British Empire Cancer
Campaign and to the United Birmingham Hospitals Endowment Research Puna
for financial support.

REFERENCES

Bielschowsky, F.— (1944) Brii. J. exp. Path., 25, 1.— (1947) Brit. vied. Bull., 4, 382.
Dao, T. L. and Gbeinek, M.— (1960) Proc. Amer. Ass. Cancer Res., 3, 103.

Idem AND Sunderland, H. — (1959) J. vat. Cancer Inst., 23, 567. ^ Ar^rshall

Folley, S. j.— ( 1952) In ‘ Marshall’s Physiology of Reproduction . Ed. Marsnaii,

F. H. A. London (Longmans), p. 546.

Geschicktek, C. F. — (1939) Science, 89, 35.

mDUCTION OF RAT BREAST TUMOURS

007

Geyee, R. P., Bleisch, V. R., Bryart, J. E., Robbins, A. N., Saseaw, I. M., and
Stare, F. J— (1951) Cancer Res., 11, 474.

Idem, Bryant, J. E., Bleisch, V. R., Peirce, E. M., and Stare, E. J.— (1953) Ihtd.,
13, 503.

Howell, J. S. — (1959) Acta Un. hit. Gancr., 15, 103.

Huggins, C., Beiziaeelii, G., and Sutton, H. — (1959) J. exp. Med., 109, 25.

Idem, Grand, L. C., and Brillantes. F. P.— (1959) Prnc. nat. Acad. Sci., Wash., 45,
1294.

Kim, U. and Furth, J. — (1960) Proc. Amer. Ass. Cancer Res., 3, 125.
ilACKENZiE, I. — (1955) Brit. J. Cancer, 9, 284.

Maechant, j. — (1958) Ibid., 12, 55. — (1959) Nature, Bond., 183, 029.

Moon, H. D., Simpson, M. E., Li, C. H. and Evans, H. M. — (1950) Cancer Res.. 10. 549.
Noble, R. L. and Walters, J. H. — (1954) Proc. Amer. Ass. Cancer Res.. 1, 35.
ScHOLLEE, J. AND Caenes, R. E. — (1958) Ibid., 2, 343.

Shay, H., Aegerter, E. A., Gruensteiy, M. and Komarov, S. A. — (1949) J. nat. Cancer
Inst., 10, 255.

Idem, FeiediSiann, B., Gruenstein, M. and Weinhouse, S.— (1950) Cancer Res., 10,
797.

Idem, Gruenstein, M., and Harris, C.— (1956) Acta Un. hit. Cancr., 12, 733.

Idem, Harris, C., and Gruenstein, M.— (1952) J. nat. Cancer Inst.. 13, 307.

Skoeyna, S. C., Ross, R. C., and Rudis, L. A.— (1951) J. exp. Med 94 1
Syimeontdis, a.— (1954) J. nat Cancer Inst., 15 539 > - •

66S

THE GEADUAL COH^H)ESION OF A SPONTANEOUS MOUSE
]\IAI\I]\rAEA^ CAECINOMA TO AN ASCITES TIBIOUR

G. BIANCIFIORI ajcd F. CASCHERA
From the Division of Cancer Research, University of Perugia, Italy

Rccoiv’ed for publiention August JS, lOGD

The capacity of tumours to convert from tlie solid to the ascitic tj^e varies,
spontaneous mammarj' adenocarcinomas in mice being regarded as amongst the
most resistant in this respect (Klein and Klein, 1955). In the present experiment
an attempt rras made to convert 9 solid mouse mammarj' carcinomas, 5 spon-
taneous and 4 transplanted, to the ascitic form.

JIATERLVIi AND METHODS

Passage of solid tumour

The mammary tumours occurred in 3 strains each carrjdng the milk factor
(Table I). When the tumours had attained a size of about 1-5 cm. in diameter
thej’’ U’ere removed and necrotic tissue u’as discarded ; the healthy tumour tissue
was Avashed in physiological saline, minced Muth scissors and strained tlirough
fine metal-wire mesh by means of a small pestle under dropping salt solution.

Table I , — Mammary Carcinomas used for Intraperitoneal Transfer in Order to

Obtain Ascites Tumours

Tumour

No.

TjiJo nnd
gonorntion
Spontaneous

Transplanted

( 1 )

Transplanted

( 1 )

Spontaneous

Histological

t-i-pe

Mucoid adeno-
carcinoma (M)
Mucoid adono-
carcinoma (M)
Polygonal cell
carcinoma (B)

Transfer
generations
attempted
. 33-37 .

Mucoid adeno-
carcinoma (M)

Transplanted . Polygonal cell
(3) carcinoma (B)

Spontaneous . Small spherical

adenocarcinoma (A)

Spontaneous . Mucoid adeno-
carcinoma (51)

Spontaneous . Small spherical

adenocarcinoma (A)

Transplanted . Small spherical
(3) adenocarcinoma (A)

51 = 5Iucoid adenocarcinoma of Olivi. Biancifiori and Barbieri (1955).
A = TjT^e A of Dunn (1953).

B = Tj^p® B of Dunn (1953). .

Figures in parentheses represent number of generations of transplanta

Fate

Ascites tumour
produced
Always solid
abandoned
Mixed fluid &
solid, transfer
by fluid which
failed to take
at gen. 13
Always solid
failed to take
Alwaj's solid
failed to take
Always solid
abandoned
Always solid
abandoned
No takes

No takes

Full name
of strain
Balb/C fostered
on C3H/CB/Se
As above

As abm'e

C3H/CB/Se

. As above

Balb/C fostered
on C3H/CB/Se
. As above

. BIII/Dm/Se

, C3H/CB/Se

COCTEBSm- OE ™>AEV CAP.CNOMA TO ASCITES TCMOCP.

The resultmg tumour suspension tvus adjnstctl to contain nliout .lO pci cent
tumour.

‘^'“Thrtot™ mo*iastic and non-neoplastic cellular

Thus an approximate estimation could he made of the percentage and actual
numbers of neoplastic cells transferred.

Experimenial procedure

The scheme of tumour transfer to successive generation.s uns as follows :
1 ml. of the fluid extract of the tumour to be tested was injected ini rapcntoneally
into 4 or 0 three-month old female mice of the strain of origin of the tumour.
The hosts were allowed to survive as long as possible in order to obtain a maximum
amount of solid tumour and peritoneal fluid for transfer to succeeding liost.s.^ If
solid tumour only resulted, tliis was suspended in the same way as the starting
material and injected into the subsequent generation. If solid tumour and fluid
was obtained, an extract of the solid part as well as the fluid were injected into
the next generation. As soon as successful transfer of the fluid portion seemed to
be established, transplantation of the solid portion ceased.

RESULTS

Development of the ascites tumour

Of the nine tested (Table I) only one spontaneous tumour converted to tlic
ascites type. The others were abandoned : (a) because the extracts of the solid
tumour failed to take ; (6) because after many generations only solid tumo\ir
resulted (tumour 2) or (c) because, having obtained successful fluid transference
for a few generations, finally no takes were obtained (tumour 3).

Fig. 1 shows the scheme of transfer to succeeding generations of tumour 1
(Table I). In the early generations both solid tumour and peritoneal fluid were
transferred, but noth the exception of the 5th, the fluid always failed to take At
tlie ninth generation and thereafter fluid took successfully, producing a dimini.siiinc
amount of solid tumour until at the 33rd-37th generation, according to tlie liim
(Fig. 1), no solid tumour was obtained. °

trai^fers of tumour No. 1 , the solid fraction was usuallv
on f) n volume and the appearance of small pin-head nodules

a bulky solid mass reaPuear^^^ ™

usually decreased. FinX the fluid tumour-cell content of the fluid

portion. ^ increased at the expense of the solid

Maintenance of the ascites tumour

time, api)ro.Sttl3^3oTa^sSs™trS Present

Sr?

670

C. BIANCIPIOBI AND F. CASHERA

Tumour -cell content of the peritoneal fluid,

A relationship between the percentage of tumour cells in the peritoneal ’
exudate and the mean survival time of the hosts was sought (Fig. 1). During the
period of conversion from solid to ascites tumour, i.e. from the start until the 30feh

6
7
6

12
13

Percentage T cells

1-5

22-9

/5-5

13-7

• 20
• 13

Survival time in days

lOOf 17
6'0(24
IOOB27
9-1 ■44
7-4 ■ 74

• Solid tumour
■ Peritoneal exudate
O Ascites tumour

22i

6SB

22 i

12-1 P

18-6 ■

S7p

IS4 ■

103 P

I8S i

IS6P

1 s ■

2Sbi

IBM

3S i

Il3p

3-si

SB i

isoi

2'3

6-Sp

2-9 i

124 i

I'7P

74 ■

3'6B

IS8 ■

1-4

■

13-7 P

11-3 i

4-2 P

17 2 i

10 3

104 P

loop

7 -3 ■

163 P

7-6 P

21 Op

bSp

17-1 P

120

■

8-3 P

183 P

7-2 P

93 i

ISO

104 H

130 P

103 P

188 P

131

■

108 B

32S p

124 ■

224 ■

II 1

128 B

3S8 p

17-3 P

27 8 ■

198

I50B

33 2 ■

190 P

32-8 P

2S6

2l'0B

3SO I

23 3 P

3S0p

274

23'S B

314 ■

2SOB

92 6 0

28 S
90-3

4 1 B
93-3 0

930 6

24 8 P
231 P

324 P

91 3 0

Fio. 1. — Scheme of transfer of solid tumour No. I or peritoneal fluid to succeeding generations
of hosts and point of conversion to ascites tumour.

TG = transfer generation

A-F = main line and sublines of transfer.

generation, there tvere great fluctuations of both factors in all the lines. Hmvever,
once coiatrersion had taken place there 33'as a dramatic rise in tumour-cell content
of the ascitic fluid and an associated decrease in host surAuval. This has been
maintained during the subsequent period of transfer of the various lines.

EXPLANATION OF PLATE

Fig. 2. — Tumour No. 1. Solid polygonal cell mammary carcinoma (type M) from Balb/C +

mouse. H. and E. X 50.

Fig. 3, — High power view of tumour seen in Fig. 2. H. and E. x400. . . r ta

Fig. 4 and 6. — Neoplastic cells (with mitotic figures) of the ascites tumour derived from o
above mammary carcinoma at the 30th transfer generation. Acetic-orcein. X 600.

British .Tnrn.v.M, or (’ani i:n.

XIV. N.i, J.

COm^EBSION OE MAMMAKY CABCINOMA TO ASCITES TUMOUB

071

DISCUSSION

Of the nine tumours tested, one spontaneous mueoid
in a Balb/C+ mouse carrying the milk factor was successfully coin cried to
aseheVtumour It may or n^ not he significant that the on y other umour
which seemed likely to convert (No. 3) arose also in tins strain tlioiigli ^

previous tumour it had been transplanted once prior to test and was of so ul
nolvaonal-cell type. The factors which determine the conversion arc virtually
unknovm, but it may weU be that tumours 6 and 7 would Iiave converted at a
later date had transfer been continued. The present tumour can rightly be called
an ascites tumour as it conforms noth Klein and Klein’s ( 1 Do.'j) definition of a tuinoui
“in which active multiplication of free neoplastic cells and/or cell coiujiloxcs
can he sho^vn to occur in the peritoneal fluid, leading to a high absolute and
relative concentration of tumor cells, that is to say. to a nearly pure cult uie
Klein and Klein (1955) tried, nithout any success, to convert 17 spontaneous
mammary adenocarcinomas to the ascites tj^pe. Thej' did, however, succeed in
converting four to six transplanted mammary adenocarcinomas. Tlicse authors
give a good summary of previous conversions of solid tumours to ascites tumour.s.

Klein and Klein (1951) found that there was an inverse relationship between
the tumour cell content of the inoculum and the survival time of tlie hosts. I’liey
suggested that “ large numbers of virulent tumor cells multiply freely in the pcii-
toneal fluid, and their ovemlielming effects kills the animals within a short time.
This short survival time prevents the formation of more voluminous solid tumor.s
In the present experiment this stage seemed to be reached at about the 3flth
transfer generation.

SUMMARY

An attempt was made to convert nine mammary carcinomas, five spontaneous
and four transplanted, occurring in three strains of mice carrying the milk factor,
to ascites tumours by repeated intraperitoneal transfer. The attempt was success-
ful in regard to a mucoid adenocarcinoma occurring spontaneously in a femalo of
the Balb/C-f strain.

The change from a combination of solid and liquid to completely liquid tumour
took place in 5 different lines at the 33rd to the 37th generation. In the generations
prece^ng the final stage there had been an increase in the tumour-cell eoiflent

All ^ tendency to decrease of the sundval time of the host

tran^L maintained by intraperitoneal

14, Maiyland. UB.A. of Health, Public Health Service, Bethesda,

fro foccivod

. m BEFERENCES

63,040. ‘

Ouv., u,,„, 0. B44.B4K, G r r

Anat. Univ. Perugia,

672

TOUS-CELL RELATIONSHIP
IN GOLDEN HA3ISTEES BY

IN KIDNEY TIBIOUES INDUCED
THE MILL HILL POLYOMA ITEUS

G. NEGRONI AND F. C. CHESTER3IAN

From the Division of Experimental Biology mid Virology,
Imperial Cancer Research Fund, Mill Hill, N.TT.T

Received for pubKcation October 4, 1960.

SIiLL HrUi potyoma virus (iVI.H.P.) ivas isolated from the spleen of a leukaemic
AK mouse in 1959 (Negroiii, Dourmashkin and Chesterman, 1939). It has many
properties in common with the Stewart and Edd}' po^'orna vims (Stewart, Eddj",
Haas and Borgese, 1957 ; Stewart, Gochenour, Borgese and Gmbbs, 1957 ;
Eddj’’, Stewart, and Touchette, 1958; Stewart and Eddy, 1959) and, further-
more, rabbit antisera, prepared against each of these viruses, cross-react in the
haemagglutination inhibition test (Negroni, unpubhshed).

High doses of M.H.P. injected into 1-5 day old hamsters produced kidney
tumours in over 90 per cent of the am’mals together with vascular lesions and/or
tumours of the liver, liearfc and lungs (Chesterman and Negroni, 1960, unpub-
lished). From the kidne}’’ tumour cells M.H.P. virus could always be re-isolated
in mouse embrjm tissue cultures, but the serial transmission of the virus from
hamster to hamster proved to be difficult. Only 5 out of 26 animals inoculated
with cell-free extracts from kidney sarcoma showed tumours after an average
incubation period of 219 days. This suggests that only a small amount of vims
is present in the tumour. The purpose of this paper is to present data which
confirm this and which clarify the cell-virus relationsliip in these kidnej^ tumours.
The kidney has been chosen in our studies because of the prevalence of tumours
in this organ, and their earfy appearance after virus moculation.

SIATEBLAL AND METHODS

Animals. — C3H mice are bred by brother-sister mating. Mice are killed on
the 14th day of pregnancy and cultures prepared from the embrj'os. Golden
hamsters are bred in a closed colony but not by brother-sister mating. Thej
are inoculated with virus 1-5 daj’^s after birth, either by subcutaneous or intra-

peritoneal route. i ■ j .

Tissue culture. — ^Mono-laj’-er tissue cultures from C3H mice or hamster kidnp
tumours are prepared in roller tubes by the method of Melnick ^

tubes are kept statioirarj’' for 1-3 days at 37° C., and then rolled at 37 C. f le
medium is 10 per cent calf serum, 0-5 per cent lactalbumin hydrolysate an
S9-5 per cent Hanks solution plus antibiotics. The medium is changed twice

Fj,.j,^._The virus is a tissue culture line passed serially with O-I ml. of medium
contaim’ng I0®-10' tissue culture infective doses (T.C.I.D.). The cultures are
inoculated within one week of setting.

Vraus-CELI. EELATIOXSmr IK KIUKI'.V TrMCH HS

S is tatoi «s the lesKliUiliei. shntvinp com, .let.' I.»e,„..wl..-

ivhibiHm M.-lV0.f„l.l eeriel '"'"'''’''“"J,''™'';™,'";
sera are challenged ^vith 8 haemagglutinatmg doses of \ / j i„,

and then left at 4° C. n’ith 3-4x10^ gu.nca-pig red blood cells. 1 1 c o<l la.int

is taken to he the last dilution preceding complete haemagglut mat lou.

InfectiviUj titralion.-T!en-MCi serial dilutions of I'”;';"’’'

groups of 7 day old tissue cultures of mouse embryo and imtdmti d at .li .
The medium is changed twice a week. The experiments are J’' '

after the inoculation. The 50 per cent end-pomt is calculated .n tlie m. liml
of Reed and Muench (1938) on the basis of cytopathic changes and haemaggliitina-

The haemagglutination test is carried out at each cliange of medium: at the
end of the experiment homogenized tissue culture cells arc addl'd to the meflimii.
The haemagglutination test was positive only when the tissue culture cells sliowed
cytopathic changes.

EXPERlJtEXTS AXD RESUI.TS

Relationship of dose to incubation period in mouse embrijn fibroblasts (MKF)

The inoculation of ilEF cultures with large amoimts of MUR
T.C.I.D.) produces cytopathic changes in the cells which hccoiiie apparent after
5 days. The period between infection and the detection of such changes increases
as the amount of virus in the inoculum decreases.

Fig. 1 shows three experiments in which the time of ayipcarancc of the cyto-
pathic changes is related to the number of infectious doses. 'J’hc results witli
high doses are uniform but the appearance of cytopathic changes is scattered
over a long period of time (2-3 weeks) Arith smaller doses.

Minces of kidney tumour cells, from hamsters inoculated with M.1I.I‘. wlien
1-5 days old, were used to infect monolayer tissue cultures of nioii.so emhrvo
cells. Qdopathic changes in these cultures were noticed in the 2nd or 3rd weeh
after infection.

Attempts to reveal additional virus masked by antibody in the tumour

changes, holevefSd Sf tata as proof ™ svlopalliie

of antibody as measured by the haemaeelutinar ^ •'>gl> litres

the results obtained in tw^ experiment! in wb’ K 3 show.s

killed at 3-day intervals after the inoculation^'^ *i<'i»i'Stcr.H were

»««« in ..ea-.oa„ Wiens, '

111

674

G. NEGEONI AND F. C. CHESTERMAN

^prus-induced hamster kidney tumours can be transplanted serially into
adult hamsters— the sera from these animals contained no antibodies to the
virus. Table I shows the results of the haemagglutination inliibition test idtb
12 sera from hamsters with transplanted tumours. The animals were chosen
trom various transplant generations between the first and the tenth. The sera

Fio. 1. — Correlation between time of appearance of C.P.E. and infecting dose of viriKS
(three separate experiments).

Time in days

Fig. 2. — Time of appearance of cytopathie effect (C.P.E.) in mouse embryo tissue cultures infected

with virus from hamster tumours.

Each square refers to one animal.

v'ere only collected from animals showing tumours at the site of implantation.
The interval between the inoculation and collection of sera varied therefore,
according to the number of the transplant generation, 280 days in the first, an
14 days in the tenth. There was, however, uniformity in the result ; tl^ sera
tested showed no antibody in the haemagglutination inhibition test. The absence
of antibodies in the sera of adult hamsters with transplanted kidney tumours,
compared with the high litres of antibodies in the sera of hamsters witii virus

vn>«S-CELL BELATroSSHir IN- KU.NNV ITMon.S

(i7a

«„crf l,mo,.rs. further indtoted lh«f those tumours ouly eouln,,,..,! n s.u.,11

“"Z expot^uf uus devised, thercrore, o h'""' ul''o/t'*,r;:;ld

be excluded. The results are sumniari/.ed in Table 11. t

Fig. 3.— Haeraagglutination inhibition titresof Berafromhnm.stcrs killctl nl intorvnlu tift.-r iiirK-iiIntini.

with virus.

5-day-old hamsters.

1 -day-old hamsters.

Table l.—Haemagglutination Inhibilion Tilres of Sera
from Hamsters with Transplanted Tumours

Transplant
generation
Control
1 .

1 .

2 .

3 .

3 ,

10 ,

Days after
inoculation

280

224

273

218

Titre

<100

676

G. NEGKONI AUD F. C. CHESTEBJIAJ^

centrifuged cells from a hamster kidney tumour was inoculated into mouse
embryo fibroblasts. These showed cytopathic changes 16 da 3 ^s later. The
supernatant from the above centrifugation, injected into mouse embrjm fibro-
blasts, produced no cytopathic effects. From the same tumours mono-layer
tissue cultures were prepared and grown in conditions identical to those used for
mouse embryo fibroblasts infected with M.H.P. When the cultures were estab-
lished the semi-confluent sheets were composed of large, fusiform cells which
were well-preserved throughout the duration of the experiment. The medium
from these cultures was serially diluted and inoculated on the 9th day into
groups of mouse embrjm fibroblasts. Cytopathic effect was only noticed in
cultures inoculated with 0-1 ml. of I in 10 dilution of medium. The cells from
the same cultures were trypsinised on the 16th day and counted under the micro-
scope. Half was added, at various cell dilutions, to mouse embryo fibroblasts.
The other half was frozen and thawed 5 times, and then inoculated into groups
of mouse embryo fibroblasts separate^ at each dilution level. Ho difference was
noted between the two groups and c 3 ’topathic effect was only found in cultures
inoculated with 3 x lO"* cells per inoculum.

Table II. — Injectivity of Virus from Hamster Kidney Tumour Cells

Hamster kidney tumours

Washed cells,
C mg.

M.E.E.»

C.P.E.

-f ve 16 days

Supernatant

Dil. C.P.E.

0 — ve

10~^ — ve

0 2/3 3 X 10> 2/3

10-1 1/3 3 X 10^ 0/3

10-= 0/3

C.P.E. = cytopathic effect.

* Mouse embryo fibroblasts,
t Frozen and thawed, or intact cells.

Kidney virus litres before the development of macroscopic tumours

Two experiments were carried out to see whether multiplication of the virus
occurred in the kidney before the tumoxu's became established. Suckling hamsters
inoculated when 5 days old in Experiment 1, and 1-day old in Experiment 2,
Avere killed at three-day intervals, from the 3rd to the 12th day. Equivalent
amounts of pooled kidney tissue, 8—32 mg. from 2—3 animals, were diluted serially ,
and 0-1 ml. was inoculated into groups of cultures of mouse fibroblasts at each
dilution level. The results are shown in Table III. Wliile a small amount of
Aurus was present throughout the period of our experiment, there was, however,
no significant difference in the amount of virus recoAmred from the kidney at any
time after the inoculation.

DISCUSSION

Virus titrations carried out -wnth tumours from hamster ki(toey shoAv that
only a small amount of virus is present in the tumour cells. This explains w y
cell-free preparations from tumours, when inoculated into hamsters, produced

VIBUS-CELL RELATIONSHIP IN KIDNEY TUMOms

(177

T«le m-hkdiula TUrts o/ Virm fmm S-n wj.
TLie 0/ nLdcr^ Kind al hicrmi. After h,on,hl,o,i

Days after
inoculation

T.C.T.D. of M.ll.l’. in
hninslor kidneys
-y —

Exi>criincnt I
1
1
1
1

Kxperiinenl 2

10--='

in’-!'

10'-=

lO'-"

Experiment 1 .l-dny-old Iiainstcrs.

Experiment 3. — 1-dny-old liamsters.

no tumours in one experiment or only a few in a second experiment after a long
incubation period. Tissue cultures established from the tumours also contam a
small amount of virus, and tliis shows clearly that antibodies arc not ma.sking
larger amounts of virus. This is also indicated by the lack of ant ibody format um
in hamsters with transplanted tumours, ilultiplication of the virus in the kidney
of the hamsters could not be demonstrated at any time after the inoculation.
It may be concluded that these tumour cells arc not virus producing ; however,
there can be no doubt that the virus is directly rcsponsililc for the jirimary changc.s
in the cells which lead to the formation of the tumour. The very early ajipcarance
of this tumour in the kidney, where tumour cells in small groups arc already
present on the third day after the inoculation in some animals, precludes any
sequence of initiation and promotion (Chesterman and Negroni, lOGO, unimb-
lished).

There are substantial differences between the results discussed in this pa])cr
and those found in cultures of mouse embrjm tissue infected with polyoma, \\dicn
tissue cultures of mouse embryo fibroblasts are infected with large amounts of
polyoma virus, virus production starts on the second da 3 ', and reaches a peak on
the fifth day after the inoculation. (Vogt and Dulbecco, 1 900 : Negroni, 1 900).
With smaller amounts of infectious virus the same peak of virus production is
obtained but after a longer incubation period. The cells which seem more
susceptible to the action of polyoma are the epithelial cells ; the fibroblasts are
apparently more resistant. If tissue cultures are infected with polj'oma virus
and then receive 24 hours later rabbit anti-polyoma antiserum the epithelial cells
die and detach from the glass smTace while the fibroblasts reconstitute the cell
sheet by migration and multiplication (Negroni, 1960). Removal of antibodies
however, is promptly followed by cji;opathic changes in these fibroblast cultures’
In similar experiments carried out with kidney cells from suckling mice the
resistant fibroblasts did not show cjdopathic changes after the removal of anti-

lodies ; moreover they did not show such changes after re-inoculation with a
second dose of virus.

The destraction of mouse epithelial cells which probably occurs as a result
of virus multiplication inside their nuclei (Banfield, Dawe and Brindlev iSo
Negroni, Dourmashkin and Chesterman, 195.9 • Dourmaslikin v
1959) does not occur in hamster kidney cells In L ^ ^^^^^roni,

fc hamster tissue and the moielssue 'ZnXZ''

produe. v,r„s. I„ the hamster, infection lead., immediate! “ o'S

678

G. NEGEONI AND F. C. CHESTERMAN

tumour ; in the mouse, the tumours arise after a longer latent period, and the
factors which determine the time of appearance of these tumours remain to be
discovered.

SUMMARY.

Mill-Hill polyoma (M.H.P.) virus produced kidney tumours in over 90 per cent
of hamsters inoculated when newly born. Virus titrations \vith mixed hamster
kidney tumours showed that only a small amount of virus was present in the
tumour cells. Tissue cultures established from the tumours also contained a
small amount of virus showing that antibodies were not masking larger amounts
of virus. No rise of antiviral antibodies was detected in the serum from hamsters
with transplanted kidney tumours. The kidney tumours may occur as early as
the 3rd day after inoculation of M.H.P. virus. This indicates that virus is
directly responsible for the primary change in the cells leading to the formation
of a tumour.

We are indebted to Dr. R. J. C. Harris for advice and encouragement, and to
Miss P. Adams and Mrs. J. E. Hadaway for technical assistance.

REFERENCES

Banfibld, W. G., Da we, C. and Brindley, D. C. — (1959) J. nai. Caricer Inst., 23, 1123.
Dourmashkin, R. R. and Negroni, G. — (1959) Exp. Cell Res., 18, 573.

Eddy, B. E., Stewart, S. E. and Touchette, R. — (1958) Proc. Amer. Ass. Cancer
Res., 2, 294.

Melnick, j. L. — (1955) Ann. N.Y. Acad. Sci., 61, 754.

Negroni, G. — (1960) Exp. Cell Res., in press.

Idem, Dourmashkin, R. R. and Chesterman, F. C. — (1959) Brit. med. J., ii. 1359.
Reed, L. J. and Muench, H. — (1938) Amer. J. Hyg., 27, 493.

Stewart, S. E. and Eddy, B. E. — (1959) ‘ Perspectives in Virology ’. Ed. M. Pollard.
New York (Wiley), p. 242.

Idem, Eddy, B. E., Haas, V. H. and Borgese, N. G. — (1957) Ann. iV.l . Acad. t>ci.,
68, 419. . „

Idem, Gochenour, A. M., Borgese, N. G. and Grubbs, G. E.— (1957) Virology, 3, SbU.
Vogt, M. and Dulbecco, R. — (1960) Proc. nat. Acad. Sci., Wash., 46, 365.

on the oncogenic activity ov Tin<:

TORONTO STRAIN OF POLYOMA VIRUS

MICHAEL STOKER

From the Medical Be-sanch Council Experimental Vimm Besenreh Unit and Dn^rlmml nj

Virology, U niverstty of Ola-egnm

Received for publication Septomber 22, lOHO

POLYOJIA virus was first isolated by Stewart and Etldy from leukao.nie iniev
(Stewart et ah, 1957) and was subsequently shown to I'bl'icc ‘"'"oiins

after inoculation into newborn mice, banisters, rats and rabbits. 1 ins virus has
been designated SE polyoma virus. A virus with siinilar iiropertic.s was siH.'-o-
quently obtained from mouse mammary carcinoma tis.siics by Mcl.iillnrh el at.
(1959). This virus is antigenically related to SE polyoma, but there is a lower
pathogenicity for mice (Howatson cf ah, 1900). For convcnienco we refer to if
as T (Toronto) polyoma. Another polyoma-like virus (MH polyoma) has also
been isolated by Negroni, Dourmashkin and Chesterman (1959), which is related
antigenically to T polyoma (Stoker and Maepherson, unpiibli.shed). It apjie.ars
from the results of Rowe et ah (19596) that strains of (lolyoma virus may bo
common commensals in laboratory mice, even in stocks with a low tumour in-
cidence.

Polyoma virus, like Rous sarcoma virus, has several advantages for inve.stigat-
ing the natm-e of viral oncogenesis. It has a short incubation period and high
efficiency of tumour induction; it grows in mouse embryo and other cells in
culture giving high titre virus stocks ; the cytopathic effect induced in mou.«e
cells permits assay by the plaque technique (Dulbccco and Freeman, Hlott ;
Winocour and Sachs, 1959). The -virus agglutinates red blood cells, so a sinqile
method is also available for enumerating total virus particles (Eddy cl ah. H>5S ;
Kahler et ah, 1959). Finally, the virus is stable under ordinary phy.siological
conditions. ‘ *

Tliis paper describes studies in dosage requirements for induction of tumours
by polyoma virus in hamsters. T polyoma virus has been used because of its
high pathogenicity for these animals.

Tissue cultures

cubated in modified Eaele’s medium -n.nfbrT,-o 4-u ^'>d m-

»cias and vitamin,. lo s "S

680

MICHAEL STOKER

Virtis stock

T polyoma virus, derived from the strain isolated by McCulloch et al. (1959)
was kindly sent from Toronto by Dr. L. Siminovitch as infected tissue culture
fluid. Virus stock (Stock 1) was prepared from monolayer cultures of mouse
embryo cells inoculated ivith either 0-01 or 0-1 plaque forming units (PITJ) per
cell. Medium was collected from cells during cytopathic changes on subsequent
days, pooled after a preliminary titration of haemagglutinin centrifuged at
250 g for 5 minutes and stored at —70°. Another stoek (Stock 7) was obtained
from virus after cloning by three cycles of plaque purification. This was con-
centrated by homogenization of infected cells in a small volume of distilled water,
and the virus was partially purified by exposing the crude homogenate to 3 cycles
of extraction rvitli fluorocarbon.

Plaque assay

The technique used was a modification of that described by Dulbecco and
Freeman (1959) and Winocour and Sachs (1959). Confluent primary or secondary
cultures of mouse embryo cells in 60 mm. Petri dishes were used. The best
secondary mono-layers were obtained when the cells from confluent primary
cultures rvere put back into the same dishes.

After removal of the medium, the appropriate dilution of virus was added in
0-2 ml. volumes to each plate. After adsorption for 2 hours at 37° C., an overlay
was added to each, comprising 6 ml. of medium with 2-5 per cent horse serum and
0-9 per cent agar (Difco Bacto). The cultures were fed with 3 ml. of the same
overlay mixture on the 4th and 8th daj^s. Neutral red O-Ol per cent was in-
corporated in the final overlay.

Plaques appeared as pale areas with diffuse borders which contained stained
and heal thy -looking cells as well as degenerate cells (Dulbecco and Freeman,
1959). Fresh plaques appeared on successive daj's, and final counts were not
made until the number remained more or less stationary, usually by the fourth
day after staining for secondary cultures, but not until the eighth day for primary
cultures.

The number of plaque forming units was determined by counts on at least
3 plates with 10 or more plaques per plate and a standard suspension of virus which
had been titrated many times and under varying conditions, was included in each
assay to check the sensitivity of the batch of cells.

Some secondary monolayer cultures underwent a spreading degeneration after
ten days or more under the overlay mixture. On affected plates tliis appeared
as a gradually increasing and sharply demarcated area of complete cell degenera-
tion, which on successive days sometimes involved a large proportion of the
culture and thus obscured the plaques. The cause of this degeneration has not
been determined. Though increased by additional neutral red, it is apparently
not due to photosensitization (Green and Opton, 1959).

H aemagglutination titrations

After heating at 37° for 15 minutes to reduce inhibitor activity, serial twoTold
dilutions of virus suspensions were made in cold phosphate buffered saline (F13b
of Dulbecco and Vogt, 1954) in 0-2 ml. volumes in perspex haemagglutmatmg
trays. To each dilution was added 0-2 ml. of cold 0-5 per cent guinea-pig erjdhro-

ONCOGENIC ACTIVITY OF POEYOMA

VlUV'S

r.si

cj-tes diluted in buffer. The snnllcst qutd^ffv^ff Vinireau^ partial

0-™ml foUmvedbv 0-2 ml. of 0-5 per cent guinea-pig erythrocyte... 1

Hire ivas taken as'the lughest dilution of antiserum .shou'ing partial inln hi mn of

hfemag^^^^^ titres of below 1/100 were taken to be due to inlnlutor

rather than antibody.

Animal inoculations

Pregnant Syrian hamsters were delivered to the laboratory froin a dealer
3-7 days before parturition. The young from the.se mothers were inoculated
24-48 hours after birtb by injection of 0-1 ml. of virus suspension, either Ultra-
abdominaUy or subcutaneously into the back. The aninial.s in each lit t er received
the same inoculum. There was no obvious variation in result., between different
litters from the same breeder receiving the same inoculum. Animals which
disappeared, mostly in the first week, and a few which died later without detect-
able tumours were not counted in the results.

Tests for polyoma antibody in the breeding ham.sters from the dealer con-
cerned have not shomi evidence of natural infection in the stock, 'riic hamsters
were observ^ed daily or tnice dailj% and wlien sick or dead were removed for post
mortem examination. Specimens were fixed in 10 per cent formal saline and
after sectioning were stained -with haematoxylin and cosin. I'hc skull contents,
spinal cord and skeletal sj'stems were not examined.

Pregnant rats were also obtained from a dealer and the iittens inoculated and
observed in the same way. Weanling Swiss mice from a dealer were inoculated
noth 0-1 ml. quantities of virus suspension for antibody production tests (Rowe
et al, 1959a). The mice were killed 21 days later and blood was collected separ-
ately from each animal for haemagglutination inhibition tests.

Tumour ’production in hamsters

Stock 1 which was used for most of the animal inoculations, contained 10®-««

. “f undiluted stock co„tai„i„g

0 PFV was inoculated mtra-abdominaliy into newborn hanistere tlieo died

— ?aSk"ceCter' ir

tes„ItofintL.abdomTnarh“l“Le ftom “ »

rl, ago and perlMps died from mial o°t hepatic Jiuto ' ""

renal tumoum, hepatic necrosis and haemSL " 'nocnlation, slimring

682

MICHAEL STOKER

With lower doses of virus from Stock 1, there was an increasing delay before
death Fig 1 shows the relationship between virus dose and the interval before
death from tumour. When or PFU were inoculated, some animals
revealed no macroscopic evidence of tumours when killed 23 weeks after
inoculation.

tumours. Open columns indicate survivors without tumours.

Types of tumour

The only lesions observed macroscopically in animals dying in the first two
weeks after intraperitoneal inoculations were those of the ludney and liver.
Detailed histological examination of other organs was not carried out however.

Hamsters inoculated with 10^-®® PFU or less, djdng in the third week or later,
developed tumours in certain other sites as well, particularly in the m3mcardium,
and in the peritoneal cavity where they appeared as multiple poljps on the
surface of the gut. More rarely, tumours were found in the anterior media-
stinum, suprarenal, testis, lung, and subcutaneous tissue. All these tumours
histologically resembled spindle cell sarcomata.

Subcutaneous instead of intraperitoneal inoculation of 10=-®® PFU resulted m
tumours of the kidney, and myocardium, and hepatic necrosis and haemorrhage.
Even though this route of inoculation was followed bj^ deaths in the third or

OKCOGENIC ACTIVITY OT POGYCMA VmCS

bloodstream. .,.„„,.r.cpnnir'allv ro«oinblod rn vcrnous

mal cells rather than vascular endothelium.

Tumours and necrosis produced by clonal stochs of rims

It rvas possible that the different tumours and more particularly the .iccnitie
lesions of the liver, might be produced by a mixture of dilTercnt. 'J”:

particle in the inoculum. Accordmgly clones of vims denved from smi, e jiart irh
we obtained by tliree consecutive plaque isolations. Two sucli clone.- nen-
respectively inoculated into hamster litters, and each produced tyiucal renal,
myocardial and gut wall tumours as well as hepatic necrosis and haemorrhage.
It therefore appears that a single virus particle carries tlie genetic potentiality
to initiate several of the different lesions.

Time of appearance of tumour cells

Since hamsters inoculated with large doses of virus tlied in less than two
weeks -with massive renal tumours, an attempt was made to detect, the first
appearance of tumour cells.

Eighteen hamsters in 3 litters were inoculated intra]ieritoiicaIly with lO'-"*'
PEU of virus Stock 1, and pairs of animals were killed subscciucntly, 2, 4. 7, tt
and 11 days after inoculation. Both kidneys from each animal were taken for
macroscopic examination. As controls, hamsters were inoculated with tissue
culture medium from uninfected mouse cells, and single animals were killed for
removal of kidneys at the same successive time intervals.

Tw'o days after inoculation of the virus no difference could be seen hetween
control and infected kidneys. Four days after inoculation however, clusters of
abnormal cells were clearly seen in the kidney's from the infected hamsters (Fig. 2
and 3). These cell clusters were in the region of the collectiiu' tubules near the
cortico medullai^^ junction. They lay in the intenstitial rciioii between the
tubules and each contamed 20 to 30 large polygonal cells with pale vesicular
nuclei jibtotic figures were present in 3 per cent of these cells, compared to
0-5 per cent of the ce ls in the mten^ening normal regions. Xo such clusters of
cells were seen m the kidneys from the control animals killed at the same or anv

2mJ age.’' additional normal hamstem of the

..ine P™-'- After

the kidneys aere eZ«d r ^ moculation.

tissue was replaced by spindlf cells (Fig ^ ^

and — teS indn.ionn

to he a featnre „t the early

684

MICHAEL STOKEE

Quantitative aspects

It is clear from Pig. 1 that the inten.’^al until death, or other obvious mani-
festation of tumours in the hamsters, is increased as the dose of Aurus is reduced.
Thus a determination of the true minimum tumour producing dose should entaii
obsenmtion for the whole life span of the hamster to detect any late developing
tumours following inoculation of limit doses. ®

This ideal was not realized, but Table I sIioavs the proportion of hamsters
developing macroscopic tumours during two periods of observation after inocula-
tion with Amrying doses of A’^irus.

Table I. — Proportion of Newborn Hamsters and Bats Subsequently Developing
Tumours, or Weanling Mice Subsequently Developing Antibody, After Inocu-
lation with Varying Doses of T Polyoma Virus

Tumours induced in :

Inocult
/ ^
Dilution
factor

ini

(PFU)

Log 10

r ^

Hamsters

, ^ 1

(C3 days) (165 daj-s)

Eats

(175 daj-s)

Antibod}'
production
in mice
(21 days)

5-88

19/19*

2/3

10^

4-88

8/8

0/8

10=

3-88

3/3

4/10

10’

2-88

3/4

18/21

0/5

8/8

10'

1-88

1/4

G/9

0/4

8/8

10’

0-88

0/5

0/6

8/8

10®

-0-88

7/7

10=

-1-88

3/8

Controlsf .

0/7

0/8

0/9

0/8

* Number of animals vith tumours/number obseri’ed for time given. Last column gives number
of weanling mice developing antibody after 21 days/number tested.

t Controls were inoculated with medium from, or with extracts of, virus free cells.

When the experiment was terminated at 63 days after inoculation, the apparent
ID50 was 10^-^® or 240 PFU. This corresponds to 340 PPU per tumour producing
dose if the Poisson equation applies. When the hamsters were left for 165 days,
however, a liigher proportion detmloped tumours. The ID60 could not be
measured but the figures suggest a tumour producing dose of 75 PPU or less.

The focal distribution of abnormal cells seen in the kidneys 4 days after
inoculation of hamsters Avith large doses of virus raised the possibility that each
focus Avas a clone of cells originating from a single initial Aurus cell interaction.
If so, the number of foci should be proportional to virus dose.

EXPLANATION OF PLATE

Fig. 2. — Tubule region of kidney from baby hamster 4 days after inoculation with lO*'®® PFU
of T polyoma virus, showing foci of abnormal cells. Haematoxylin and eosin x 75. _

Fig. 3. — Tubule region of kidney from normal hamster of same age as that shown in Fig.

for comparison. Haematoxylin and eosin X 75. .

Fig. 4. — Part of medulla and cortex of kidney from baby hamster 11 da 3 's after inoculation
with 10®-®* PFU of T polj'oma virus, showing replacement of medulla bj’ spindle cells.
Haematoxj’lin and eosin x75. , . , .vi

Fig. S. — Single focus of tumour cells in kidnej’from baby hamster 9 daj'S after inoculation wi n
J 02.88 PFU of T polj'oma virus. Haeraatoxj’lin and eosin x 150.

0>'C0GEX1C ACTIVITY OV I>Ol,YOMA YIIU'S

r.s'i

By assuming that the foci were distrihutccl evenly tlirovi^ghout the
it .vas estimated from counts of foci in a single secUon of each Ju<hu > . Ih.
approximately 103 foci per kidney resulted ,

more accurate counts, 12 hamsters ^vere each nmculated

10 hamsters we inoculated u'ith PhL . 1 hey were all Killed e.tlu r .*

or 10 days later, and both kidneys from eacii annua! were fixed and .‘=enall\
sectioned. (Only one kidney was obtained from each of two hamslen; inoculated
with 103-33 PFU. These two single kidneys have been taken as coining from
one animal, maldng a total of 11 kidney pairs in the hatch.) . r • r

The serial sections were examined with a dissecting microscope and foci of
tumour cells easily recogirized and counted (Fig. a). The diaincter of the foci
were 100-200 /( and were such that examination of a total of :10 or mt>re n'giilarly
spaced sections tlu-ough each kidney was sunicient.

The results shown in Table II show a correlation between numbens of foci and
dose of virus at the two dilutions tested and agree well with the rough e.'itiiiiate of
103 foci per kidney after inoculation with PFU (Ratio I’FC/focus r57.'»)-

The observed frequency distribution of foci in the grouji inoculated with KG '"
PFU (Table III) agrees best nith the theoretical distribiitioii for an awrage of
1-7 focus forming particles per dose, calculated according to the Poisson formula,
or 440 PFU per renal focus.

Virus

Inoculum

I ^ \

Dilu- PFU

tion Log

factor 10

Iff' 2-88

10 ’ 1-88

Xuinher of foci in pairs of kiiii)cy.<!

00 00 01 01 01 02 02 12 22 22 24

00 00 00 00 00 00 00 00 00 02 — .

irith Indiratn]

Do'ir of

.M.-.'in

HntRi

jK'r

riav

nuiinnl

forijH

24 .

. ^40

2 . im;

. Cl 1 r»

Taule IIL~Frequency Distribulion of JRenal Foci per Anmal ('ompornl
Theoretical Distribution from Poisson Equation

dumber of renal foci per nniinnl

Hamsters with number of renal foci
shown

Observed distribution
Theoretical distribution for 1-7
focus forming units per inoculum

0-18 0-27

0-18 0-31

0-18

0-20

O-O!)

0-1.7

0-18

o-or,

O-OI

0 - 0.0

:o-oi

CO-01

owS' fo‘f6aX' to '-0

Table shows that the number finallw ob ■ cells at 9_lo days

686

MICHAEL STOKER

several months, it also suggests that tumour cells do not metastasize in the
unafFeeted kidney.

Table W. — Comparison of Number of Kidneys Involved in Each Inocnlated
Hamster at 9—10 Hays and in Subsequent Observation Period

Inoculum
^

Renal foci at 9-10
days in :

Renal tumours presenfc
afc 165 days or
earlier death

Dilution

PFU

One

Both

r- >

One Both

factor

log 10

^^one

kidney

kidneys

None kidney kidneys

10’

2-88

6 7 4

10’

1-88

! 9

7 2 0

Further evidence against metastases comes from the small total number of
tumours found in the various organs of each animal after small doses of virus.
Table V gives the frequency distribution of macroscopic tumours in all organs
examined. It shows that the number is roughljj^ related to dose of tous and
that most animals dying after inoculation of a small quantity of virus onl}^
developed a single tumour. It should be made clear, however, that a detailed
liistological search ivas not made, nor were the skull contents, spinal cord, or
bones examined.

Table V. — Frequency Distribution Shotoing Number of Tumours Observed Macro-
scopically at All Sites Examined in Hamsters Inoculated with T Polyoma
Virus

Inoculum

C ■■■ \

DUution PFU

factor log 10

10 ’ 2-88

I0< 1-88

Number of tumours
per animal

» ,

0 1 2 3 4 5

3 4 7 3 2 0

3 5 1 0 0 0

These results suggest that a single particle of virus can initiate a focus of
tumom’ cells in the Iddney, and that this will probably occur if some 300-500
PFU are inoculated intra-abdominally. It should be noted that the dose required
to produce a tumour in any site is not much less, and this suggests a relatively
high susceptibility of the renal tissue.

Tumour production in rats

When 24—48 hour old rats were inoculated with T polyoma virus, they deve-
loped renal tumours resembling those seen in hamsters. More rarely tumours
rvere present in lungs, heart and abdominal wall. From the proportion of rats
wdth tumours showm in Table I it would appear that these am'mals are less suscep-
tible and unsatisfactory for titration of oncogenic activity.

Mouse antibody production test

Rowe et al. (1959a) have showm that weanling mice develop haemagglutiiun
inhibiting antibodies if inoculated with small amounts of rurus and thus maj^ be

0S7

on-cocekic activity or roi.voMA viuvr

W results of a titration of stock 1 ..l.t-nucl

and confirm the high of virus' From the iiropnrt inn |)f>'dliv(’

through omission of a sufficiently lou - f •• , , ,„iiiimuiii ainniint

in the group, given a mean of 0-0 lo 1 1 u per i
required to produce antibodies is apparently about n 1., 1 I I .

DISCUSSION'

The observation of very early tumour development after nmvhnri. Imm^tor.
are inoculated u-ith undiluted suspensions of T polyoma mhis ngrep it
reports of McCulloch et al. (1950), Axelrad el al. (1000) and Ham r! al. {\. - ).
Not only do the animals die with massive renal tumours 1-2 ivei'ks after moeiil.i-
tion but foci of large abnormal cells u-ith a high mitotic index can b(> .‘■■.•(m between
the developing tubules of the kidney as early as 4 days after mociilat mn.

In the series reported in tliis paper, the appearance of the tumniir cells was
not preceded by ohdous degenerative changes in the interstitial ecdls between the
tubules as described by Ham ef al. (1060) but such changes may have Ix-en
missed by infrequent sampling. Marked ccntrilobular haemorrhagie neerou'^
of the Uver was indeed present however, in all animals exiiosed to high ilo^c--
of virus, and death was often due to haemorhage from the characteristic blood
filled cysts in the liver.

As might he expected reduction in virus dosage greatly delays the deatli or
other obvious manifestation of tumour formation and this interferes with titra-
tions for oncogenic activity. The focal distribution of the early renal lesions
suggested that each group of tumour cells might be a clone initiated by a single
virus particle. Though numbers are small, the counts of foci showed a fair
agreement with virus dose, and, at high dilution, an approximation to Poissemian
distribution. This relationship would not apply if some of the foci were initiated
by -sdrus which multiplied in the animal after inoculation, nor if tumour eidls
had formed secondarj^ foci. The relatively constant size of the foci at 0 davs
also implies a nearly simultaneous origin.

suggested by these results, the foci are clones of tumour cells, each
imtiated by infection with a single virus particle, one must ask if it is possible
for the foci to reach the size observed 4 days after infection. Counts on 12 foci
showed an average of 26 ceUs visible in the sections. Assuming that foci are
spheres and that the sections are approximately equatorial, the number of cells
|) r focus can be calculated from the section thickness and size of the cells result inn

ceU rfdalf ' V- « divisions from a siimit

cells in viw k'Z ta Ctat iThoL“ -^^ 00 ^ 8 ^ 0 ^^ r""’}''

cells.

688

MICHAEL STOKEE

From the results, it appears that the foci of tumour cells do not retrogress.
Despite the extremely active sarcoma-like grovdh of the early tumour cells
however, the frequency of single tumours in animals dying man}^ montlis after
inoculation with minimum doses of vims suggests that the tumours do not meta-
stasize. If so, the multiple tumours developing after large doses of virus are
due to primary infection of the different sites, rather than secondar}’^ migration
of tumour cells.

Though one virus particle can probably initiate a tumour, it is clear that many
partieles must be inoculated before tliis is likely to occur. Whether or not ail
particles are competent to induce tumours is not knoum, but there would obviously
be a large wastage, quite apart from back leakage of the inoculum, since suscep-
tible cells apparently occur in only a limited number of tissues. In terms of plaque-
forming particles, several hundred must be inoculated to initiate a tumour in the
kidney, but less than a hundred may be sufficient to start a tumour at anj^ site
(though appearance of such a tumour may take 5 months). Less than one
plaque-forming particle is sufficient to infect a mouse so as to produce antibodies,
and thougli more laborious and less accurate than plaque assay, this is still the
most sensitive form of infectivity titration. None of these methods of titration
measures total physical particles, however. From the haeraagglutinin titres and
the large numbers of particles seen by electron microscopy of viral suspensions
(Wildy et al., 1960) it is obvious that the number of phj^sical particles which
constitute the minimum oncogenic dose must be several orders of magnitude
higher than the number suggested by infectivity titrations.

Rowe et al. (1959<7) have already dranm attention to the difficulties involved
in accurate measurement of the oncogenic properties of polyoma virus. Renal
tumour focus counts as described here may provide an improved, though laborious
method of titration, but are still subject to the many variables inlierent in in vivo
sj'stems.

It is to be hoped that the virus induced transformation of cells in vitro described
by Vogt and Dulbecco (1960) and Sachs and Medina (1960), will now provide
the controlled conditions necessary for quantitative studies at the cellular level.

STOEUAKY

Studies were carried out on the oncogenic activity of the Toronto strain of
polyoma virus in hamsters. Foci of tumour cells appeared in the kidneys of new-
born hamsters 4 days after inoculation with 10®-®® PFU. The animals died with
massive renal spindle cell sarcomata and hepatic necrosis in 1—2 weeks. Reducing
the dose of virus delayed the deaths and permitted development of tumours in
heart and peritoneum and more rarely in suprarenals, lungs and testes and
subcutaneous tissue. Inoculation of cloned virus showed that potentiality to
form tumours in many sites and to cause hepatic necrosis is inherent in a single
virus particle.

Counts of foci of tumour cells in the kidneys were roughly proportional to
the virus dose and suggested that each focus constituted a clone of tumour cells
initiated by a single virus cell interaction. Inoculation of about 400 plaque-
forming units of virus was necessary to initiate one tumour focus in the kidney.
Despite the early rapid invasion of the kidney there was no evidence that the
tumours formed metastases.

ONCOGENIC ACTIVITY OE roi.YOMA Vlltl'S

r.M'

I am very grateful to Professor Capi)Pll for his general mlviee ami h.s lul,.
uith raicrophofegraphy, to Mr. Norman Russell for preimratmn of the urns
and to J\lr. J. M. McCorquodale, also for microphotogrnphy. I'lnally ni\ ihanK--
are due to Mr. Mhlliam House for his skilled a.ssistnnre in many st ages of I Ins work ,

REFEREXCKS

Axelead, a. a., McCulloch, E. A.. Ho\vat.son. A. K.. Ham. A. W. am. Simim.vit. n.

L.— (1960) J. nal. Cancer Inst.. 24, 109.").

Dulbecco, R. axd Freeman, G.— (19.59) Yirolojij. 8. IlOH.

Idem AND Vogt, M. — (1954) •/. e.rp. Med., 99, 107.

Eddy, B. E., Roive, W. P., H^artley, J. W.. Stewart. S. E. am. lluEiiMat. R. .1.
(1958) Virology, 6, 290.

Green, R. H. and Opton, E. M. — (1959) Pror. Soc. erp. hiol. S.Y.. 102. .570.

Haji, a. W., McCulloch, E. A., Axelrad, A. A.. Siminovitch. I., am. Howatsos,
A. F. — (1960) J. nat. Cancer Inst., 24, 1112.

Howatson, a. F., McCulloch, E. A., Almeida. .1. 1)., SiMiNovm n. I,.. .Vxe.i.um..

A. A., AND Ham, A. "W.— (1960) Ibid., 24, 1121.

Kahler, H., Roew, 5V. P., Lloyd, B. .L, and Hartley. .1. \V.-ql0.50) Ibid.. 22, 0J7.
McCulloch, E. A., Howatson, A. F., Si.MiNoviTrii, L.. Axia.RAD. A . A. am. Ham.
A. W. — (1959) Nature, 183, 1,535.

Negroni, G., Dourmashkin, R., and Chesterman. 1'. C.— (19.50) Ilrit. m/d..! ii 12.5'i
Rowe, tV. P., Hartley, J. 5V., Estes, .1. H. and HnaiNEii. H. ,1.- (10,59^;)’ d fx , ',
Med., 109, 379. ''

Idem, Hartley, J. 5V., Laav, L. W. and Huerner. R. ,T.— (19.595) Ibid.. 109 119
Sachs, L. Medusa, D. — (1060) Nature (iii j)rcss)

Wildy^P.^tokeh, M. 0. P., MicniEBsos, I. A. aeu Honsi;, ]!.

WINOCOUR, E. AND Sachs, L.— (1959) Ibid., 8 397
^ ogt,.M. and Dulbecco, R.-(1900) Proc. nal. Acad. Sci, Wa-d,.. 46. 26,5.

690

A HISTOCHEMICAL STUDY OF THE EARLY STAGES OF
CARCINOGENESIS IN RAT LR^R ; LOCALIZATION OF
FLUORESCENT CARCINOGEN AND CHANGES IN SUCCINIC
DEHYDROGENASE ACTIVITY

LUCILLE BITENSKY, R. W. BALDWIN and J. CHAYEN

From the Department of Pathology, Royal College of Surgeons, Lincoln’s Inn Fields,

London, W.C.2., and the

Cancer Research Department, The University, Nottingham

Received for publication August 16, 1960

There are two major theories concerning liver carcinogenesis induced by
azo-dj'es. According to tiie first, the protein-deletion hypothesis (Jliller and
Jliller, 1955) the dye becomes bound to a soluble cjdoplasmic protein wliich is then
“ lost ”, in that it is not found in the tumour cells. The second (Elson and
Hoch-Ligetti, 1945 ; Elson, 195S) holds that the more important phenomenon is
the damage to the Krebs C3’^cle produced by metabolites that inhibit succinic
dehydrogenase.

It seemed of interest to test the localization and effect on succinic dehydro-
genase of a structural!}^ similar compound, 4-dimethyIaminostilbene, wliich also
produces cholangiomata when injected into rats (Elson, 1952). The advantage
of this carcinogen is that it is naturally intensely fluorescent, so that if the whole
molecule became bound to a cytoplasmic protein, as in the first theory, it might
be seen even in very low concentration by direct fluorescence microscopy. More-
over, by the use of the cryostat microtome, the determination of succinic dehydro-
genase activity has been made semi-quantitative, as checked by corresponding
estimation on larger pieces of tissue by means of the Thurnberg tube method.
Thus both hypotheses are open to direct histochemical investigation.

MATERIALS AND METHODS

1 . Animals and diet

Albino Wistar male rats about 150 g. in weight were injected Muth 4-dimethy!-
aminostilbene. They, and control animals, were fed on a specially prepared dLt
containing 5 per cent protein and an additional 0*4 per cent methionine (Diet C .
Elson, 1952). Some rats, designated “normal animals ” were fed on a well-
balanced diet (M.R.O. rat diet B.41).

They were killed by placing them under a funnel through which nitrogen was
passed from a cylinder at a rate of over 1 litre per minute.

2. The carcinogen

The 4-dimethylaminostilbene was s}mthesized by R. W. Baldwin ; it ^
dull yellow flaky crystalline substance, with a melting point between 1

WCAUSATIOS OF CAIICINOOKS IS FAT I.IVV.U
ur 0 It tvas used as a 1 per cent solution in araelus oil. I In' Mnotio.'l (

tnuk of 4-dimet\iylamiHostilbcno is as follows .

CH,

-cm f'li-

CH,

3. Injection and dose ... , , • i

(al Intraveritoneal—T\\ei normal and the control nnnnals (destyimled aininn^

4 - aid Tl rSed no injections. One of the other rats was nijeeleri ,„tra
peritoneally wdth 0-5 ml. of arachis oil alone and was Uilleil half an hour lab’r.
The remaig animals were given 0-5 ml. of the I per cent soln mn ..f
aminostilbene by intraperitoneal injection and they were killed at mteriaU of
1 hour, 1 hour, 2 hours, 6 hours and 24 hours after the tune of injeetion : tln-y
wiU be referred to as Aj, A^, An, A^ and A,, respect iveh . ....

(b) Intravenous . — Some animals were anac.st hef ized i>y injecting intrap'Ti-
toneally 1 ml. of a 2 per cent solution of Xuinal lloche. liec.'iu.«e a good ainvav
is essential with this anaesthetic, a tracheotomy was i>erforined. An ineiMoii
w'as made in the trachea and a plastic cannula tied in position. 'I'lie port.il
vein w'as exposed and a plastic cannula was ligatured into jiosition ; the t-di-
methylarainostilbene (1 ml. of the 1 per cent solution in arachis oil) was injected
into the cannula, and the animal was killed 4 minutes later.

4. Technique of section cutting

A sample of each of three lobes of the liver was taken and frozen iinincdiately
at between —40° and —70° C. Sections were cut at S // on a freezing eryost.m
microtome at about —20° C. Details of this procedure, which is similar to that
used by Coons, Leduc and Kaplan (1951) ivill be discussed elsewhere.

The sections were dried over pho.sphorus pentoxidc in an evacuated dessicator
for 1 hour at 30° C. and then for 1 day at 0°C., except when used in studies
on succinic dehydrogenase activity, where they were stained iininediatelv.

.7. Histochemical methods

(a) Fluorescence mtcroscopi/.-Freshly prepared frozen sections were examined
by fluorescence microscopy for the presence of the fluorescent carcinogen 'riu-

"S'" "•»'

ulu* S"; fddi'S ciotTf

aminostilbene in acetone nnti/nrecinitation "^S* of dulimcthyj.

examined by fluorescence

O OS M phosphate buffer (Sor^srfs) al S 7 8 t -V f

succinate at a concentration of 0-05 m 'Mit-rn’ fidded sodium

eolutmn before use. The control inmrbation soS rcLdte at

692

LUCILLE BITEKSKY, B. W. BALDWIN AND J. CHAYBN

method, on frozen sections, gave semi-quantitative results, depending on the
time required to yield appreciable colour, or to produce the same amount of
colour as a standard. After they were stained, the sections were mounted directly
in Farrant’s medium.

(c) Janus Green. — Freshly prepared frozen sections were mounted in a 0-01
per cent solution of Janus Green in 0-85 per cent sodium chloride.

(d) Oil Red 0 . — Freshly prepared frozen sections were fixed for 5 minutes in
a 10 per cent solution of neutralized formalin (40 per cent Formaldehyde) con-
taining 0-9 per cent sodium chloride. The3'^ were rinsed in distilled wafer, im-
mersed in 60 per cent iso-propjd alcohol for 3 minutes and then in the Oil Red 0
solution (saturated Oil Red 0 in isoprop3'l alcohol 3 parts, distilled water 2 parts)
for 10 minutes. The3’' were rinsed in 60 per cent isoprop3d alcohol and then in
distilled water, counterstained with Harris’ haematox5din and mounted in
Farrant’s medium.

RESULTS

1 . Observations by fluorescence microscopy

(a) Normal and control livers. — Normal and control liver sections exliibited a
faint autofluorescence ; the liver cells were ver3’^ pale green with brighter green
outlines and dark non-fluorescent nuclei.

(b) Liver sections from injected animals. — ^The carcinogen, dissolved in aracliis
oil, emitted a strong blue fluorescence under ultraviolet light — arachis oil itself
was non-fluorescent.

Liver sections from am'mais treated with intraperitoneal injections of 4-di-
meth3daniinostilbene showed no increase of fluorescence. In order to ensure that
the carcinogen was reaching the liver, it was injected directh’^ into the portal vein
in some animals. Hflien sections of the liver were examined, no characteristic
fluorescence was seen.

(c) “ Staining ” of normal and control liver sections by 4-dimetltylamifiostilbe?ie.
When normal and control liver sections were examined b3’’ fluorescence microscop}'
after “ staining ” with an aqueous suspension of d-dimetlndaminostilbene, the
entire section exhibited the bright blue fluoi’escence of the carcinogen. This
fluorescence faded uniforml3^ and after 5 minutes the sections showed 03113' normal
autofluorescence.

(d) “ Staining ” of liver sections from injected animals by i-dimethylamino-

stilbene. — Although sections of the livers of animals injected with the carcinogen
did not show fluorescence due to the aim’nostilbene, the}' could be ‘'stained
b3' immersion in an aqueous suspension of 4-dimeth3'laminostilbene ; as with
the control sections, this induced fluorescence faded within 5 minutes. In
addition, “ stained ” sections from the animal killed half an hour after injectioii
showed bright blue fluorescent droplets in the periportal cells, which retaine
the fluorescence for 15 minutes, that is, for 10 minutes after the generalize
fluorescence had faded. It could be demonstrated b}' staining with Oil Reel u
that these were fat droplets. _ .

(e) To test ivlietlier fading of the fluorescence of i-dimethyhnnijiostilbene was nie
to quenching by cellular constituents. — ^Normal liver sections, fixed b}' forma m,
and then “ stained ” in an aqueous suspension of 4-dimeth3'laminostilbene, ex
hibited generalized bright blue fluorescence. On repeated examination 3
fluorescence microscop}', this fluorescence was constant and did not fade.

LOCALISATION OV CAIUMNOOKN IN HAT LIVV-U

Despite tills result, it Avas apjiarcnl that the fading of the lluoreseenee could
be due to quenching by some unknown cellular constituent which is niivct i\ ated n
formalin. However, in an animal which died under aiiae.sthctic. and which uas
injected intravenously into the portal vein immediately after death, the fluores-
cence of the carcinogen was seen strongly throughout the smusoid.s and a .so in
the cjdoplasm of the liver cells in frozen sections. Hence, where it might he
expected that metabolic activity, particularly oxidation, had i.een stoiiped. no
fading occurred.

2. Histochemical eslinutlioti of .‘fucctuir. (Irhijdrofjaiiixc ortirili/

Succinic dehydrogenase activity was estimated hy the rate of ajipcarance and
intensity of red stained grannies in the cells. In the livens of animals Aj,- and Ac,
cjToplasmic granules were coloured red after half an hour s incnhat ion. with very
intense staining after 2 hours. In animal Aj no stain was observed after half an
hour and only faint coloration after 2 hours’ incubation. 'I’lie staining tif animals
Aj and A, was stronger than Aj but still weaker relative to Aj,-. 'flic cytoplasinic
granules in the liver cells of A^, and A^j. however, .stained as did the normal, that
is the succinic dehydrogenase activity was recovered 0 hours after injection.

To assess the relative activity in A», .sections of Aj were incubated for 2 hours
and the intensity of reaction seemed identical with that seen in sections of A^,
incubated for onl}’ 1 hour. Hence the succinic deliydrogena.se activity in Aj
was reduced hy approximately one half.

3. Janus Green staining

Since succinic dehydrogenase is t\-picaliy a mitochondrial enzyme, it was
advisable to see how far the changes in the activity of this enzyme could be cor-
related unth morphological alteration. For this purpose, sections were stained
with Janus Green. Mitochondria were seen in various forms ; filaments, rods,
granules and globules could be identified, all of which stained selectively with this
dye.

Filainentous mitochondria were found only in the normal and control livers.
After injection of carcinogen, the mitochondria were in the form of short rods
and granules. Globular forms were also seen in some of the liver cells and per-
sis ed in the livers of the animals killed 6 and 24 hours after injection. In addition
green staining droplets were seen in the cjdoplasm of the periportal liver cells
Oil V f killed half an hour after injection. These droplets stained with
in they consisted of fat. Fat droplets were also present

ini ^ h^^Portal cells of the liver sections from the animal killed 1 hour after
them ™ animal killed 2 hours after injection, but

after inject^” droplets m the sections from the animals killed 6 and 24 hours

1 - Fluorescence

addtd 4-dimethylaminostilbene, no

mathylaminosthbenr mS. The blue fluorescence of 4-di-

fluorescence mav W ^ “^sked by the normal autofluorescence or ds
may be quenched, or the carcinogen may be rapidly met^bofizS

694

LUCILLE BITENSKY, K. W. BALDWIN AND J. CHAYEN

and tlie fluorescence destroyed. When a solution of 4-dimetliylaminostilbene is
viewed under ultraviolet light, the blue fluorescence is constant and does not
fade, showing that it is not destroyed by ultraviolet light. Moreover, when
sections are “ stained ” by a suspension of the carcinogen, blue fluorescence is
visible and is distinct from the autofluorescence. Hence, if the fluorescence of
the carcinogen were present, it ivould have been seen and therefore almost cer-
tainly ivas not masked by the autofluorescence.

Snapper et al. (1951) found that the fluorescence of stilbamidine was quenched
by nucleic acids. That this is the explanation of the results reported in the
present communieation is unlikely for trvo reasons. Firstly, no fading occurred
when a section, fix'ed bj'- formalin, was “ stained ” by a suspension of 4-dimethyl-
aminostilbene ; it is possible, however, that some unknomi cellular constituent,
responsible presumably for quenching, had been inactivated by formalin.
Secondly^ examination of a section, taken from an animal which had died under
anaesthetic and which was injected intravenously into the portal vein immediatelj"
after death, showed brilliant fluorescence of the carcinogen which persisted and
was not quenched ; in tliis case metabolic activity, particularly oxidation, had
presumably been stopped and no fading of fluorescence was observed.

Thus it appears likely that the fading of the fluorescence that occurs after
“ staining ” with the carcinogen, and the lack of fluorescence in the injected
animals is due to rapid metabolism of the carcinogen to a non-fluorescent form.
Where the “ stain ” is taken up preferentially by fat droplets in the liver cells,
the fluorescence is destro3’’ed less rapidl3'^, fading after about 15 minutes, and this
delay is probably due to the partial protection against metabolism afforded to the
carcinogen by the fat droplets.

2. Succinic dehydrogenase activity and Janus Green results

After injection of 4-dimeth3daminostiIbene, the mitochondria lose their fila-
mentous form and appear as rods and granules. Similarly, in the animal killed
half an hour after injection, succinic dehydrogenase activity is much reduced.
In the animals killed at longer intervals after injection, however, although the
mitochondria still appear damaged morphologically, the enzymie activity has beeji
restored to the normal level.

At first sight, these observations could be interpreted simply as follow’s .
the fact that the mitochondria can be deformed grossly and yet stain well for
the enzy'^me suggests that their morphological integrity is not essential for succinic
dehydrogenase activity. Hence the reduction in the activity of tliis enzyme
shortly after injection of the carcinogen implies a direct inhibition of the enzyme.
Such an inhibition has been demonstrated by Elson (1952, 1958) for oxidize
metabolites of the related carcinogen dimethylaminoazobenzene. If this sugges-
tion is correct and if oxidized metabolites of 4-dimethylaminostilbene are prescu
half an hour after injection, then it is not surprising that little or no fluorescence
due to the intact molecule, has been observed in the liver cells.

The speed of metabolism of the 4-dimethylaminostilbene might appear exces-
sively fast but such a rapid metabolism of the related dimethylaminoazobenzene
has been demonstrated by Mueller and Miller (1948), who showed that deme ly a
tion began at 10 minutes and was almost complete at 30 minutes. It is o
that the final proof that 4-dimethydanunostilbene is metabolized depends on i
chemical investigations which are now being undertaken.

LOCALISATION 01 ’

CAlunNOOKX

IN 1!AT LIVKl!

CONCLVSIONS

\fter a single injection of 4-(UmctliylaininostilluMU'. neillicr i)rnlein Iniirliin; not
permanent depression in succinic dcliydrogciiasc activity can hr . ('inmi'-tra M
Fn the liver cells. It seems clear, horvcvcr, tlmt the liver cells react sliarply to th-
carcinogen. Further injections of the carcinogen may modify I his mil lal react mn

SU.MMAUY

1. The localization of the strongly tluorcsccnt carcinogen t-dimethylamimt-
stilhene rvas studied by fluorescence microscojiy in frozen sections of rat liver.
No characteristic fluorescence vas scon after inlrajieritoiieal or inlravenou..
injection of the stilbene. The fluorescence faded rapidly in frozen liver sr-rtiom-
“stained” rvith 4-dimethylaminostill)enc, but persisted in dead or li\e<l liver
sections. These results appeared to indicate rajiid metabolism of the rareinogen
and there was no evidence that the whole molecule became bouinl in the livi-r
cells.

2. Morphological damage to the mitochondria of the liver ec'lls appeared soon
after injection and persisted for at least 24 hours. 'I’lierc wa*; eoneoniilani
depression of succinic dehj'drogeuase activity soon after injection, but thi-
recovered to normal levels in a few hours, 'it appeared likelv that siieeinir
dehydrogenase was directly inhibited, probably by metabolites of the earrimigen.

REFERENCES

BaLDWTO, K W^, Bes™, J., CH.VYEN, J. and CrNNINOHAM. C. lilllO) ,|r/„ f,..

Coons, A. H,, Leduc, E. H. AND Kafl.an. N. H — (lO.ill./ rj-n oa i— >
Elson.L.A.— ( 1952)Rn7. J. Caacer, 6 . 3 !» 3 .— wrj'lluU ”14 in'"

AIillER, E. 0. AND illLLEE, J A T iiri( n r . 4

ATttt'ttx.t, nr- --r ’ Cancer /a.vf., 15 , If, 7]

Muetter P P t 15. l.oTl

’

din. Med., 3T562: ’ •• E.-

(I it'd)./.

696

A HISTOCHEMICAL STUDY OF THE EARLY STAGES OP
CARCINOGENESIS IN RAT LIVER : CHANGES IN CELLULAR
LIPIDS AND MITOCHONDRIA

LUCILLE BITENSKY, K. W. BALDWIN aud J. CHAYEN

From the Department of Pathology, Royal College of Surgeons, Lincoln's Inn Fields,

London, ir.C.2, and the

Cancer Research Department, The University, Nottingham

jReceived for publication August 16, 1960.

Cholangxojiata occur in a high proportion of rats tJiat have been injected
intraperitoneally with 4-dimeth3daminostilbene, particularly if the}" are fed on
a diet containing a “low concentration ” of protein (EJson, 1952). This carcino-
gen is particularly useful for investigations of the mechanism of tumour induction
since it emits a blue fluorescence when activated by ultra violet h'ght, thus allowing
its intracellular distribution to be observed by fluorescence microscopy. Such
studies shoM'ed, hoivever, that the fluorescence due to the presence of 4-dimeth3d-
aminostilbene in cells of the rat liver faded rapidly (Bitensky, Baldwin and Cha 3 "en,
1960). This might have been due either to metabolic changes in the molecule
Of to the speedy excretion of the carcinogen. The following is a report of histo-
chemical investigations made to elucidate the immediate response of the lirer
to the carcinogen.

JIATEHIALS AlfB METHODS

Materials and methods are as described in the preceding paper (Bitensky,
Baldvdn and Chayen, 1960).

Histochemical methods

(a) Acid haematein method (Baker, 1946). — Freshly prepared frozen sections
Avere fixed in the dichromate mordant at 22° C. for one da}" and then at 60° C.
for one day. They were rinsed in distilled u'ater, immersed in the solution con-
taining acid haematein for 5 hours at 37° C. and then in the borax -ferricyanide
differentiating solution for 18 hours at 37° C. The sections were rinsed, de-
hydrated and mounted in Canada Balsam.

A positive reaction with the acid haematein method is thought to indicate
the presence of phospholipid (Baker, 1946 ; Chayen, Gahan and La Cour, 1959).
It was difficult to record accurately the great variation of intensity of the aci^
haematem reaction. This was overcome by the use of Kodak “ Microdak
panchromatic 35 mm. film which was found to give a sufficiently linear response
over the wide range of light intensities encountered. This film requires specia
handling and processing, and reference should be made to the manufacturer s
instructions. The yellovdsh non-specific background produced by the aci
haematein method was suppressed by the use of a magenta colour filter (Vr^

No. 35). Thus, to produce accurate representation of the various intensities o

CHAXCtES IX CKLIA’EAU Ul’ins AXU MIToCltnXlM'.l A

!.' i 7

reaction with this staining technique, each slide wns p!n>l'>grn|ili>'>i lu nia^'rnia
light with a standard exposure and under standanl ojiliral e<indilinn- AH Uif
subsequent photographic procedures wen* ecpially st.andardircd. 'rin’ " Mirru
dak” film ensured that all the cxposnre.s gave a jiroporiinn.iio p!i>i!<v;fAp’iii
response.

(h) Extraction by mcthanol-chlorqform.—Vn'i^hW pn-panil fru.-i-it <
immersed in 1:2 (v/v) methanol-chloroform for } hour.-; n1 22 C, ■ri!r\ urr,-
then treated mth the acid hacmatcin method ns described abm e,

(c) Prolonged acid haanatcin method. — ^'I'o denmn'.trale the men- tudith
bound phospholipids, freshly prepared frozen sections wen- fixed in the dl-),re|.i •.•'e
mordant for tliree days at G0“ C. (see “ Di.scn.vsion ”). 'I’hev uetr the„ -rd
by the acid haematein procedure as dc-scrihed above

Sections xvere stained xvith Oil Red 0 and danns Cn.-n ns nire.viv de .
(Bitenskj^ Baldxx-in and Chaycn, 1000 ).

Oil Redo i-sn.Ts

Oil Red 0 stained neither normal nor control liver t ■

hyer sections taken from the animal infected wit I?.. i . T'
of the animal injected intravenouslv and l-;ii n ' In tli.- lur-t

buted at random contained stainedYatdronfrts 'n detn-

killed half an hour after intraperitoneal inieef nnim-d

periportaUatty change uith a Lall cmitrSrn^ ^»>..ued ,,

(Fig. 1 ). Iir the fats killed iXofl S
change gradually diminished in extent fFie ti.e fr..,

killed 24 hours after injection li^.T of

flecks which stained u-ith Oil Red 0 were ollbotml, s„,,n

-■ Mitochondria

u . ^

IV,

icid haematein (Iwg. si.

698

LUCILLE BITENSKY, R. W. BALDWIN AND J. CHAA'EN

In the livers of the injected animals there were some cells that stained more
darlily, hut unlike the random arrangement of such cells in the control, they
showed a definite centrilobular distribution. Twenty-four hours after injection
recovery seemed to have begun in that the cells stained bronm, although not as
strongly as did the controls, and the distribution of the darker-staining cells had
reverted to random.

4, Acid /laematein staining after methanol-chloroform

In the normal and control livers, the vessels and sinusoids stained blue, as
did the cytoplasm of a few of the liver cells. The remaining liver cells were
coloured broivn and the mitochondria w'ere still visible. After injection of the
carcinogen, a similar blue reaction was seen in the vessels and sinusoids of the
liver, but in the animals killed up to 24 hours after injection, none of the liver
cells stained blue. The periportal cells remained almost unstained, while in
the centrilobular cells, the cytoplasm was coloured pale grey. In the liver of the
rat killed 24 hours after injection, a few of the liver cells showed the blue staining
and the cytoplasm of the remainder appeared bronn in colour.

5. Prolonged acid haemafein method

After treatment for tliree daj's in the hot dichromate, the c 3 doplasm of the
liver cells in the normal animal stained dark brown, w'hile the mitochondria were
blue-black. The intensity of the stain was increased in the control livers, how-
ever, and w'as greater still in the liver from the animal killed 24 hours after
injection. A similar enhanced staining Avas also seen in the centrilobular areas
of the liver from the animals lulled up to 6 hours after injection, but the periportal
cells remained unstained and appeared emptj'.

DISCUSSION

1. Results with Oil Red 0 and with the acid haematein procedure

Oil Red 0 is simply a colorant of fats, that is, it colours regions contaimng
a high concentration of lipoidal matter, but like manj^ “ fat stains ” it does not
demonstrate highlj’^ dispersed or hj’^dropliilic lipids (Berg, 1951 ; Chaj^en et ah,
1959). The acid haematein reaction, on the other hand, is considered to show
the presence of phospholipids. Although Baker (1946) believed that it needed
to be controlled by his pyridine extraction test, this has now been found to be
unnecessary and even, occasionally, misleading (Chaj^enet al., 1959). This was
due, in part, to the effect of heat used in the extraction test, rendering certain
bound phospholipids more av^ailable for the acid haematein reaction.
quently, in the present stud}"^ tests for such bound phospholipids w'ere made by
prolonged treatment with hot dichromate, so that the dichromate mordante
the phospholipids immediately the heat freed them (Ghayen, unpublished data).
Hence, a positive reaction with the acid haematein method Aidll be considered as
indicating the presence of phospholipids.

In these studies, twm types of phospholipid could be distinguished, namej,
that present in the mitochondria and that dispersed in the cytoplasm.
though the morphology of the mitochondria Avas much altered, the intmsity o
their staining wdth the acid haematein method did not change appreciably au i

MlTOf’IH'NIitUA

('HANOKS IN

CKLIA-UAH lArn>S AKll

(’.!!!»

700

LUCILLE BITENSKY, B. W. BALDWIN AND J. CHAYEN

time after injection of the carcinogen and hence this type of phospholipid has
probably not contributed to the changes seen in the cj’toplasm.

The perplexing feature of the results (Table I) is the fact that 'within 4 minutes
of intravenous injection, or half an hour of intraperitoneal injection, there was
a striking apparent loss of cj'-toplasmic phospholipid -with concomitant increase
in free fat. It -svas shown that this was not due to deposition of arachis oil into
the liver. Moreover, it was improbable that it was caused by two distinct
mechanisms, one depositing fat in the liver and the other removing phospholipid,
all acting ’^^uthin such a short time. This is almost ruled out of consideration,
because no phospholipid was observed either leaving the liver cells, or in the
bile canaliculi, so soon after injection. It is also unlikely, in the short time after
injection, that the freed phospholipid was undergoing enzymic removal of its
phosphate moiet}'’ and being deposited as fatty droplets, but this possibility can-
not be discarded without more biochemical, and preferably isotopic, studies.

It seems more likety that the carcinogen caused a change of phase, altering
a “ fat-in-water ” into a “ water-in-fat ” emulsion. It is envisaged, therefore,
that in the normal liver cells, the cytoplasmic phospholipid is tightly bound and
occurs with its h 3 'drophobic groups away from, and its hj'drophilic groups out-
wards to, the watery cell sap. In the animals fed plentifully ivith protein, these
hydrophilic groups maj'^ be protected from the usual acid haematein procedure
by protein ; in the rats fed on a diet deficient in protein, this protection is not
present and hence the phospholipids are more available to the acid haematein
reaction. The carcinogen seems to strip the phospholipids from the protein (see
below) and to leave the phospholipid in a reversed condition, namely with its
fatty moieties outermost, and run together in droplets which are gross enough to
colour with Oil Red 0. In consequence, the hj'dropliilie (phosphate or rather
phosphatidyl choline, etc.) groups become turned inwards and therefore are no
longer available to the acid haematein reaction.

This is a complex hj'pothesis, but one that can be tested in the biochemical
studies that are now in progress.

EXPLANATION OF PLATES

Fig. 1. — ^Rat liver ^ hour after injection of 4-DAS ; phase contrast iUumination, ’

to show droplets of free fat, which appear as wliite spheres (cf. Fig. 4 for phospholipi
content).

Fig. 2. — Rat liver 2 hours after injection; phase contrast illumination, X500; the fat
droplets already are less obvious than in Fig. 1 (cf. Fig. 5).

Fig. 3. — ^Rat liver 6 hours after injection ; phase contrast illumination, X500; there is jnactie-
ally no free fat and recovery is almost complete (cf. Fig. G for similar recovery of phospno ipi
content).

Fig. 4. — Rat liver J hour after injection ; acid haematein method, x 500 ; note the

of phospholipid, i.e. the paleness of the stain, in contrast to the high fat content o
similar section in Fig. 1.

Fig. 5. — Rat liver 2 hours after injection ; acid haematein method, X 500.

Fig. 6.— Rat liver 6 hours after injection ; acid haematein method, XSOO;

content is at least half-recovered, as shown by the darker staining of the cells, while the
fat content is almost completely suppressed (Fig. 3).

Fig. 7. — Normal rat liver cells with filamentous mitochondria ; acid haematin method, X ’

Fig. 8. — Damaged mitochondria in rat liver cells 4 hour after injection of 4-D.4S , aci
haematein method, x 1680 .

CHAXGKS TX CEGU’hAn I.IJMDS AND MITOCIIOXDIUA

701

■2. Effect of vKthnnol-chlowform

When liver scctio.^s arc s,.bjecto,l to ext met inn by a .nixturo
and chloroform, the former teiub to break bi^icl-pmtem bonds. If tb( • ^
complete, phospholipid is freed and extrnetod from the see ion. If.
thehberation is incomplete, the ])reviously bound iiliosiibobind beeomes a\ada il

for stainiimbv the acid hacmatein reaction. , .t i

In the normal and control livers, whieli bud been treated wit li met Imnol-
clilorofonn, serum lipids were partially liberated from ]irotein and so s .imt i i lu
\vitliin the blood vessels ; this effect was also seen in a few of the Ii\ or ee s. * o
loss of staining occurred and therefore no phospholipid was freed .snflieient \ to
be extracted.

3. Effect of 4:-dimethiilnminostilbcnr. on lipoprotein hindinij

The almost complete removal of jihospholipid by melhanol-eliloroform acting
on sections of the liver from animals killed u]) to 0 hours after injection, suggests
that the lipids were less tightly bound than in the control cells. '1 hat- this is a
specific effect on the endoplasmic phospholipid.s was demonstrated by the fact
that the serum phospholipids were unaffected and behaved like those of the
control animals. Twenty-four houns after injection, the binding seemed to have*
reverted to the tj'iie found in the control.

It cannot be claimed that the results obtained after tlie jirolonged treatment
with dichromate mordant (.3 days at 60° C.) demonstrate the total jihospholipid
content of these cells, but they give some indication of the more closely bonnd
as well as the free phospholipids. It seems likely therefore that there is very much
less staining for phospholipid in most of the cells up to 6 houns after injection
than there is in the control. The exception to this is the cells with centrilobular
distribution, wliich appear to contain even more jihospholipid than do the control
cells, suggesting that the carcinogen lias accentuated the unmasking effect of the
hot mordant. This view seems more likely than that there is a real increase
m the lipid content, because of the speed with which the effect is jiroduced.

i. Mitochondria

Filamentous mitochondria were seen in the liver cells of the normal and control
aramals. After injection of 4-dimethylaminostilbene, however, the mitochondria
appeared as granules and short rods, but globular forms were also present cspeci-
a y after 6 and 24 hours. The damage to the mitochondria in the animals
reated mth carcinogen appears to be a direct effect of 4-dimethylaminostiIbeno

5. The general effect of the carcinogen on liver cells

although the carcinogen cannot be localized within
after inTectiou fluorescence nor by any histological abnormality so soon

chanaei I’l ’ P^®®^nce can be inferred from the histochemical and crdoloaical

702

LUCILLE BITENSKY. R. W. BALDWIN AND J. CHAYEN

SUJmARY

The immediate response of rat liver to the carcinogen, 4-dimethyiaminostil-
bene, was studied by histochemical methods on frozen sections. There was a
considerable increase in free lipid, correlated -tvith an apparent loss of phospholipid
in some of the liver cells soon after injection ; recover 3 ^ occurred gradual!}' vnthin
twenty-four hours. Other liver cells showed an apparent increase in total phos-
pholipid content. These results suggested that the carcinogen, or a metabolite,
had freed the bonds between lipids and proteins, and caused some t}'pe of phase
inversion. In addition, morphological damage to the mitochondria of the liver
cells was confirmed.

We wish to acknowledge our gratitude to Professor G. J. Cunningham, who
initiated this v'ork and who has given much encoiu’ageraent and guidance. We
are also very indebted to j\Ir. A. A. Silcox for his skilful assistance and to Mr. A.
L. E. Barron for all the photography.

We are indebted to the &itish Empire Cancer Campaign for financial assis-
tance and one of us (Lucille Bitensky) wishes to thank the Trustees of the Prophit
Fund for a research studentship.

REFERENCES

Baker, J. R. — (1940) Qiuirf. J. micr. 6’ci., 87, 441.

Berg, N. 0. — (1951) Acta path, microbiol. sca7td.. Supp. 90.

Bitensky. L.. Baldvtn, R. iV. and Chayen, J. — (1960) Brit. J. Cancer, 14, 690. ^
Chayen, j., Gahan, P. E. and La Couk, L. F. — (1959) Quart. J. micr. Sci.. 100, 325.
Elson. L. A. — (1952) Brit. J. Cancer. 6. 393.

ENVIRONMENTAL KHEE EAOU'AL^^

M. .1. LYONS* AND .1. H. Sl’KNCE
Cancer Research De]Kirtma,(, RoijtiinnHsoi) Moiiarial Ho'^pilnl. (llas.jmr

Kcccivod for iniMirntion Octolior M. lltl’iO

In work related to tlic caupation of luiinnD lung eaiicer. sonio invest igalors.
appreciating the proposed special cancer environment, of the cigarette sinolvcr.
seek to support tliis proposition by discovery of new carcinogenic agents or
adjuvant factors in cigarette smoke, to wlncli non-smokei’s are not. especially
exposed. Thus smoke phenols (Roe, Salamnn and f'ohcn. Ulo!)) and bases
(Wynder and Wight. Ifl.oT) have been cited as co-factors in wlmt would essentially
be carcinogenesis by smoke polycyclic hydrocarbons, 'riie discovery of
appreciable quantities of free radicals in cigarette smoke (Lyons. tJibson and
In^am, 1958) led to the proposal of their possible involvement in the carcinogenic
process. This followed from various suggestions in the literature on the possibh*
pivotal role of free radical forms in cancer production by a variety of agents
(for reference see Bamford and Jenkins. 1900). A more speculative aspect,
perhaps, of the free radical hjqiothesis is the po.ssiblc sensitization of cells to
the deleterious action of carcinogenic agents by free radicals through their (lara-
magnetism, an idea related to that of Howard, Hawes and Gray (I!).")!)) who are
seeking to establish whether the well-known radiosensitizing action of o.vygcn
and nitric oxide is due to the paramagnetic property of these agents.

For present purposes it was decided to attempt an estimation of the relative
amounts of free radicals to which cigarette smokers and non-smokers are liable
to be exposed, and to investigate any qualitative differences among such radicafs
wliich might have a bearing on their possible biological action. As regards the
Ltter point, it seemed desirable to have an estimate of their size and stability,
tor these ends, the following experimental work was undertaken.

EXPERIMENTAL

Free Radicals in general atmospheric pollution

Suspected sources of general atmospheric free radicals examined were;
ornestic chimney smoke, vehicular exhausts and cigarette side-stream smoke.

camples of particulate exhaust products from mechanically sound petrol
n diesel enges were collected in glass containers which were kept cool during

samples ivere collected at speeds of 2080 1400
(Npfri; overload from vehicle engines on a test bed

enmW^°*^ 4 . produced at reduced loads.) The netrol

coUected during acceleration-deceleration and idlmg pLiods
over ^ o'«^ed cars. All the exhaust condensates were filtered and dried

o^er phosphorus pentoxide. These samples were tested for the prSenS of fee

of Preventive Medicine. Sloan-Kettering Institute,

704

M. J. LYONS AND J, B. SPENCE

radicals by the electron spin resonance (e.s.r.) method, as were samples of domestic
chimney soot and general atmospheric soot — the latter collected over a two-
month period in autumn in a ventilator shaft at a site in central Glasgow.

In view of the well-known conditional carcinogenicity of soot-borne carcinogens
(Steiner, 1954 ; Nau, Neal and Stembridge, 1958 ; Von Haam, Titus, Caplan and
Shinowora, 1958) it was of interest to know the proportion of soot radicals likely
to be available to the cells of the respiratory tract-— as far as this can be loiovm
by extraction with organic solvents. Consequently, the radical levels of the
soots were re-determined following the extraction of aliquots -with hexane, benzene
and acetone. The results were as follows :

(1) The domestic cliimney soots contain 5 x 10^® free electrons per gram
of soot. Tills concentration was not affected by the extracting solvents, hexane,
benzene and acetone, whicli removed 6-6, 27-5 and 41-0 per cent by weight of
material, respectively.

(2) The diesel soots contained 1-8 x 10*® free electrons per gram, irrespective
of running condition. This level was also unaffected by the extractions, wliich
resulted in a loss by v'eight of 2, 6 and 7 per cent.

(3) Both petrol exhaust samples exhibited an extremely large broad resonance
signal (of the order of 1000 gauss) which was unaffected by any of the solvents
used.

(4) The ventilator soot also exhibited a large broad resonance.

In the case of the petrol and ventilator soots it has been suggested (B. T. Allen,
personal communication) that the origin of tlie large broad resonance may lie
in the presence of lead and iron in the samples. The free electron concentrations
were not available at the time of writing. The domestic soot samples exhibit
normal free radical absorption similar to carbon blades, while the diesel soot
absorption is broader, although still centred around g = 2. The broader absorp-
tion signal of the diesel soot relative to the domestic soot is related to its formation
at a higher temperature, and is due to the interaction of the free electrons with
electrons in the conduction bands (Austen, Ingram and Tapley, 1958).

Cigarette side-stream smoke was found to contain approximately 5 X 10**
free electrons per gram. Main-stream smoke had a “ stable ” radical concentra-
tion about tAvice as high.

Free radicals in cigarette main-stream smoke

Cigarette main-stream smoke, condensed at liquid oxj'gen temperatures showed
the presence of 6 X 10*® free electrons per gram. On w'arming to room tempera-
ture or above, or trapping the smoke in benzene at room temperature a residua
concentration of about 10*® was found. A majority of these were found to e
light sensitive, as the following experiments indicate. ,

Druckrey and Schmahl (1955) shorved that the fluorescence intensity o
freshly prepared benzene solutions of cigarette smoke decreased on exposure o
light. Jolmston (1957) showed that the labile fluorescent components were pro-
ducts of combustion of the tobacco ; that similar labile material could be produce
by combusting other vegetable matter ; that the percentage of the total fluores
cent material which was labile varied for the various material combusted.

In the present investigation it was found that the reactivity of fresh benzene
solutions of cigarette smoke with the stable free radical scavenger aa -dipnenj

KNVlKOKMV'.NTAli VUKV. UAlMt'AI.S

/? n;or\’lhvdra 7 vl (DPrH) dccrcnscd »m cxposinii tlic lornn'r t<> litlld . 1 1”**

.0 oLnr .von wl,.,, n,,- V'!”™-

alkali and organic solvents prior to sinnkmp. ^ I ' , ,

Sfe smcSvcrc foumlno! lo l,o light II «»s .h■.■..l...l In .■si vl ..lli.T

ricreSc in Itnon-sccncc inlonsily nn.l in l>l'PII-.u-l,v,ly ...T,- ,nl,.l.-.l l.h.-n..

mena and if so, to atteinjit- an cxplaniilinn. , , r it • k,, ■

DPPH-reacting polar reducing substances were extracted frttm the eiga e
prior to smoking bv renuxing witb dilute alkali, aeid and water. After eon.
ditioning the tobacco to about a Id per cent water content be tobacco wa.
smoked in cigarette form. The smoke from .me such ‘■'L"'«''He in -> " "'j- "I
benzene afforded a convenient solution for both fluorescence and DI 1 M-aeli\il,\

TUBflSlirCTllGlTt S

Solutions were irradiated at a distance of if* cm. with an unliltered high
pressure 125 W mercury vajtour lamp, giving a band sjteetnnn. and sainj'les
withdrawn at hourly intervals for measurement, h luoresccnce niea‘iuicinent s
were made on a Uvi.spck spectrophotometer with a 11 7.1(t lluorcscence at laebnient .
which afforded excitation at 30.7 in//. An d-1 mg. per e.ml .piinine sul])hafe in
01 X . H,S0^ standard was used. DPPH-activity was estimated from oliscrving
the rate of decrease of the compound's 720 m// alisorplion nmxiinum following
the addition of Od ml. of a 10~- M solution to Id ml. .if the smok.' solution, I he
percentage decrease in both cases was correlated with concentration by rt'ferenct'
to dilution cun'es for the same solution.

A remarkably close correspondence between the deer(‘as(! in llnon’scen.’e
intensity and DPPH-acti\ity was found. This is shown in ]'ig. 1.

An analysis of the cun'cs shows them to he discontinuons at Iw.i points, the
four- and six-hour irradiation points. A decrease of about, (id per cent in the
concentration of labile components occurred in 24 bom's, the major jiortion (7d
per cent) occurring in the first seven hours of irradiation. 'I'lie reaction ha.l
first order kinetics, and tlirce different rate constants revealed the presence .if
three different components. Electron .spin resonance measurements .if a sm.ilce
solution irradiated under the same conditions for two and six hours .showed that
decreases in free radical concentration of 20 and 40 per cent rcsiieclivelv Im.l
occurred.

The light sensitive components of cigarette smoke apjiearcd t herefore to he
ree radicals, a light titration of which revealed the presence of at least thre<>
species of different stability.

In order to obtain some estimate of the size of such radicals, a concentrated
ei^ene solution of smoke was added to fresh alumina (Spence, 100-200 nieslil
allowed to evaporate off in the air. The resulting alumina wifli’
S “o Care was taton througl,o4 to 0^,^ c

leaienc at*!"*' eluted, and the eamo procedure carried out with

tluee oi 3-cetone consecutively. The solvents were evaporated from rim

e , ; free radical content of the resulting torrestii W

e-s.r. The results are shown in Table I. ^ estimated by

hentr? were detected in the hexane soluble fraction while tOe o, i

P 35 per cent and iTper ^nt of

from the alum.ua noth a faefflty which increased srith the potri™of t

706

M. J. LYONS AND J. B. SPENCE

Table I. Chromatographic Behaviour of Cigarette Smoke Free Radicals

Elitaie % EhUed

n-Hexnne , 0

Benzene . 35

Acetone . 0

solvent. The experiment indicates that some free electrons are trapped in
mdividual aromatic structures containing as little as, perhaps, 4 or 5 condensed
nuclei. Such structures, it is thought, could readily gain access to the cell.

u.v. irradiation (hours)

Fig. 1. — Light titration of photo-labile material of cigarette smoke.

X X DPPH reaction .

O O Fluorescence.

^ A e.s.r. measure of free radicals.

DISCUSSION

The mechanism of free radical production in the carbonization of various
organic materials proposed by Austen, Ingram and Tapley (1958) can, it is thought,
be advanced to explain radical production from liquid fuels (in the present case,
diesel and petrol fuels) as well as domestic fuels. These authors state that
radical concentration grows when the carbon atoms begin to cluster in condensed
ring systems, suggesting that the essential mechanism in the trapping and stabili-
zation of the unpaired electrons is the existence of ring clusters possessing a lu'gh
degree of resonance energy available for stabilization. It was assumed that the
radicals were formed by breakage of bonds around the edge of carbon clusters,
but they state, the possibility of their arising from defects in ring packing is

V.NNMUONMKNTAl. ntKK KADlf.M.S

707

conceivable— tivt' ami w'vcn inemlu'ml riniiis jmHliu-injt iiilmiml (nvatrnl enrlmn

atoms.

The prc.=:cnt work ^lunv.s that the ra^lumls. in the dieM’l mh.Is nntl <I..ine>.tie
chimney soot at least, are similar to those in earhon hlaehs in Iieinj; e\ft'einel}
stable and insoluble. They jiosse.'^s a very sitnilar ahsor|i( ion line.

Their common jirojicrty. i.e. their ratlieal content, is nnit^^paired I>y I'Xtraetion
nith organic solvents, lint since extraetod earhon idaehs nre tion-e.'in’itiogf'iiie
in contradistinction to the nnexlraeted materials whieli an* fiminenlly nelive,

it is anticipated that the ty]H' of free radieal eontained in tiu'se soots is non-
carcinogcnic.

In a previous experiment (Lyons. (Jihson and Ingram. lltriS)a henzc'iie extrat'1
of general atmospheric soot wa.s found to have' a radieal netivity of ajtjiroximalely
10'' free electrons ]icr gram. This was an f)ver-estimal<' since (he extrael
contained transition elements. 'Die extrael annmnt<'d (o -<• per eeiil of tli(>
whole soob by weight. On the basis of Waller's figures (I'.iA'i) ahoul l-ld g. of
soot is inspired by the “ standard man " jier annnm. Aeei'pt ing t lie enneenlrat ion
10'' as a maxinnnn. and assuming that little nrn-st of the soot jiarl iele.s oeenrs anti
that the full radical load is available to the cells, an animal exposure t<i approxi-
mately 29 X 10’^ free electrons occurs. A .snmUer of '.W eigaretle.s jier day
can have a yearly exposure of about lh7(' x Ud’’ free electrons (on tiie basis of
the smoker receiving 30 nig. of (dry) smoko per cigarette), i.e. about (>S times the
amount received by the non-smoker. 'The radicals in (he heir/.t'iit' (‘Xlracl of the
atmospheric soot were not light -.scimitivc (in so far ns this could he ascertained
from reaction with DPPH), i.e. were of a higher order of stability.

Calculations based on such comjiarativcly slnhie atmospheric free radicals
can give a false picture however, since it is known that a jirofnsinn of active
•^ort -lived free radicals are present in polluted urban and indii.strial atmospheres,
jlney have their origin in solar-initiated chemical events involving nlijiliatic
yoroearbons emitted into the atmosphere incidental to liieir use ns litpiid fuels,
'w ^ events include the direct pliotoly.sis of aliphatic aklchydes and

e ones and the reaction of photocliemicany produced ozone with olefins. Some
egree of stabilization of these extremely reactive radicals occur.s it is thought.
} reaction with nitric oxide to form a complex (Saltzman, llloS) and pcrhajis
re certain aromatic polycyclic hydrocarbons (authors' unpublished

a “ The total concentration of these free radicals in urban air may reach
gf ^ per cent of the molecular contaminants or alternatively, a steady
(Job pressure of free radicals of 0-3 to 3-0 parts per hundred million

pool"of°f ’ portion of the total atmospheric

Tiirth° f ^ '^e-dicals carries greatest potential lung cancer hazard for humans.

remni^^ • required, Meanw'hile, tlie smoker-non-smoker balance sheet

“Wins incomplete.

O cr JL

engine soots, domestic chimney soot, and a sample of general
of electrL^ presence of free radicals using the metliod

Por gram anTs TST'/ Concentration levels of 1-8 x 10'® free electrons
resnectivAU, ^ ^e/g. were obtamed for diesel soots and chimnev snnf

ooimentratfon compHcating effect of transition elements, the Radical

on of the petrol and atmospheric soots was not available at the time

708

M. J. LYONS AND J. B. SPENCE

of Avriting. It was sliowoi that in the case of the diesel, petrol and cliimney soots,
the radicals were extremely stable and insoluble. It seems unlikely that tlie}-
play any part in carcinogenesis.

The majority of the radicals trapped at room temperature in cigarette “ tars ”,
in contradistinction to those of the soots, Avere found to be light sensitiA'e, of a
loAA^er order of stability, and of a size range which would permit their findino’
access to the cell.

Other aethm free radicals, produced in polluted urban and industrial atmo-
spheres by the action of sunlight on aliphatic material released into the air as
a result of the incomplete combustion of liquid fuels, are briefly discussed, and
their possible hazard for man indicated.

The authors are extremely grateful to ]\Ir. B. T. Allen, of Professor Ligram’s
Department, UniA'ersity College of North Staffordshire, for the e.s.r. data.

REFERENCES

Austen, D. E. G., Ingbaai, D. J. E. and Tapley, J. G. — (1958) Tram. Faraday Soc.,
54, 400.

Bameoed, G. H. and Jenktns, A. D. — (1960) ‘ Formation and Trapping of Free
Radicals ’, p. 468. Edited by Bass and Brioda, Nbav York and London (Academic
Press).

Deuckery, H. and Schmahl, D. — (1955) Science, 122, 421.

Hoaa'ARD, a., Haaves, C. and Gray, L. H. — (1959) Hep. Brit. Einp. Cancer Gampgn, 37,
200 .

Johnston, H. S. — (1956) Industr. Engng Ghem. [Industr.), 48, 1488.

Johnston, H. — (1957) Nature, 180, 1350.

Lyons, M. J., Gibson, J. F. and Ingram, D. J. E. — (1958) Ibid., 181, 1003.

Nau, G. a., Neal, J. and Stenbeidge, Y. — (1958) A.M.A. Arch. Ind. Health, 11, 21.
Roe, F. j. 0., Salaman, M. H. and Cohen, J.— (1959) Brit. J. Cancer, 13, 623.
Saltzjian, B. E. — (1958) Ind. Eng. Ghem., 50, 677.

Steiner, P. E. — (1954) Cancer Res., 14, 103.

Von Haaiai, E., Titus, H. L., Caplan, I. and Shinoayora, G. ¥.—(1958) Proc. Soc.
Exptl. Biol., 98, 95.

Waller, R. E. — (1952) Brit. J. Cancer, 6, 8.

Wyndeb, E. L. and Wright, G. — (1957) Cancer, 10, 255.

THE :^IYELOMA (JLOHUEIXS

P. HOUdHTON' ANi> X. n. MAUTIX

From Ihc Bcv«rl„n„ :} I'l.n.M Ih.-.I.M >UM FM.

IjmnoHs .1

Korciwd for pubUmtion (V'to\v4T S, llMiH

In- 1935 Mcfarlaiic clcinonM rated, in the .‘^era <>f a j.atient .Mim-ri iL fi< i
myelomatosis, a protein having an unusual sedimeulatmu enimlant. ’I '

shmvn that these sedimentation constants vary according to I lie Pf'*''
ttation at Nvhich they arc estimated (Xeurath and Pailey P.t.i.t). If Ihi're is not
gross asvmmetrv of the molecule examined, and this is the case for most seiiiin
proteins', the observed .sedimentation is a linear function of concentration, and
the sedimentation constant obtained by extrapolation to -/.ero coiiccnl ration is
related to the observed sedimentation constant by the simple eipiation .

S'jn = S^n 4- he

Sjo being the observed sedimentation constant :

S° 2 o the sedimentation constant extrapolated to zero :

c the concentration of protein in grains per inf> ml. at which the observa-
tion was made.

Koenig and Pederson (1950) have obtained K values of O-O.o and 0-‘2.') lor
fibrinogen and gamma globulin respectively. Wliilc Taylor (1952) obtained a K
value of 0-235 for albumin. It will be .seen that libriiiogen, the molecule with the
highest degree of as 3 mimetrj% has the highest K value.

McConnell and Martin (1958), in the coiir.se of a jihy.sicocliemical stiidr* of
globulins isolated from individual patients suffering from niN'cloinatosis. produced
data on the relation of sedimentation to the concentration of t he isolat cd jirodiict s.
Studies on isolated globulins have continued in this laboratory. Fig. 1 shows some
of this accumulated data in condensed form.

IITen the K values of these proteins are plotted against tlicir ciccfroplioretic
mobility, the appearance of the graphical results (Fig. 2) indicated tliat increasing
as3'mmetry is associated with increasing negative net clmrge. These denionstrahle
P h3'sicochemical changes point to overall changes in the configuration of the
globulin molecule, but they do not point to precisely defined groups of globulins
so rmch as a spectrum of molecules xs-ith a gradation of change,

Wallenius, Trautmann, Kunkel and Franklin (1957)
xvhb 1 V components present in normal iiuinan scrum

ultraSZgafrairsis®''”^^

twj aia-tTrS ; S'""-, '"'"r

710

P. HOUGHTON AND N. H. MARTIN

production of proteins present, in trace amounts, in normal sera, a possibility
considered by Slater, Ward and Kunkel (1955). ^

If, as some nTiters harm maintained, these myeloma globulins are inherently
different from normally produced gamma globulins, it is important to know to
what extent and in what way they differ. To extend the study of these globulins
it was thought that the immunoelectrophoretic technique of Williams and Grabar
modified for microanalysis by Scheidigger (1955), might yield useful information!

6 - 0 !

v\/\

/vr.

0-5 I'O

g./ 100 ml.

Fig. I. — Sedimentation concentration ratio

• • normal gamma globulin

O O myeloma gamma globulins

Each line represents the plot of the slope calculated from not less than five individual
measurements.

MATERIAL AND METHODS

In these investigations fresh serum from fasting patients, in whom a diagntwis
of myelomatosis had been established both on clinical and histological grounds,
was used. Electrophoretic analyses were made in the Tiselius apparatus using
0-2 M phosphate buffer at pH 8-0 at OT. Ancillary electrophoretic investigations,
when the material was insufficient for the classical technique, were made on paper
by the method described by Franglen, Martin and Treherne (1955). Immuno-

MVKl.OMA (JhOUri.lXS

711

electrophoretic analyses were nindo by 1 lie niicrolecliiii(|uo by Sebcubpger

(1955), and nitrogen estimations were done by the Miero-Kjeldabl met not .

Preparation of antisern

Antisera ivcrc prepared in rabbits by two met bods ; ■ ■ ,

(1) Intranuiscnlar injection of a suspension of an alumina jn-eeipitalo the
antigenic protein, according to the method of I’room (l!Md) as used b^ Ueilz
(1952).

Six injections of 10 ml. of the snsjionsion in a divided dose were gnen at. ten
day intervals. The titre started to ri.se after the second injection and bad reached
a satisfactor}’ level after the fifth injection.

(2) Suhentaneons inoculation of the antigenic protein using 1' reunde s adjuvant .

2-5r

.o-o

•j - «

"S’

Sl-5

ShO

0-25

0-5

‘K’

0-7o

Fio. 2.— Electrophoretic mobility of normnl nnd myeloma globulin.^ plotted ngaitiHt K valiie.-!
derived from concentration sedimentation studie.s.

• normal globulin
O myeloma globulins

The following antisera were used in these investigations :

(1) Antisera against the freshly drama scrum of two patients known to be
sanenng from myelomatosis, designated G and K, and showing anomalous protein
patterns in the beta and gamma areas respectively.

(2) Antisera prepared against the G4 fraction of pooled normal sera containinc
^r cent gamma globulin.

no f original G4 globulin used for the production of antibodies contained

globulins demonstrable by classical electrophoretic anabasis
of analysis against normal sera suggested the presence of a trace

g buhn having the mobility of alpha globulin (see Eig. 4, third column).

RESULTS

Table I shows the ti
buhn content of the fi

serum proteins and the alpha, beta and gamma elo-
sera subjected to immrmochemical analysis These

712

P. HOUGHTON AND N. H. MARTIN

values were obtained by electrophoretic analysis in the classical Tiselius apparatus
Kepresentatjve Schlieren patterns are sho%m in Fig. 3.

The inimunoelectrophoretic analyses against the tliree specific antisera are
sho^TO in Fig. 4. Serial dilution studies of these proteins against the antisera did
notlung more than emphasize the constant characteristics of the patterns obtained.

Serum 'W’ Serum ’G’

Fit!. 3. — Pilot ograplis of Tiselius’ analysis of serum “ AV and serum “ G ” at 21 and 3 hours.
Analysis of a 2 g./lOO ml. protein solution in 0-2 molar phosphate buffer at pH 8-0. at O’C.
and at a potential gradient of Ov/cin.

Table I. — Serum Proteins and the Alpha, Beta and Gamma Content of the
Five Sera Subjected to Immunochemical Analysis.

% Total of components
(globulins)

Patient

.. „

Total piotem
g/100 ml.

Alb.

alpha

. 5

beta gammaj

7 —

gammao

“ R ” .

. 5

T ” .

G 60

“ G ” .

7-S

. 7

C” .

9!)

DISCUSSION

The physicochemical data referred to in the introduction suggest a progressive
deviation in the structure of the myeloma from the normal globulins rather than
a clear cut differentiation. Tliirteen out of nineteen end group analyses of myeloma
globulins listed bj^ Putnam (1956) showed aspartjd or glutamjd or both to be
present. These are the end groups commonlj’ found in anal 3 ’sis of gamma globulin.
The carbohj’drate analj’sis of jMiiller-Eberhard and Kunkel (1956) and of McConnell
and Martin (1958) showed variations in content from values within the normal range
to values more than ten times the upper limit of the normal range.

MYKI.OMA CLOIiri.I.VS

7 I .-5

Differentiations based on iinnuinoloixirnl sludies stem linek to tlio work of
Bajiie-Jones and Wilson (1022) and Hobinstm (I!)27). 'Hk' oarlif'st worloTs in flic
field concluded that tlie tnyeloina jirofeins won* not a sin^di* cnlily but a ^ronp
differing one from another. Tins lias been largely eonlinneii by more rerent work
by Wuhrmann. Wnnderlcy and Hassig (ItCiO). who .'.fn‘s>ed the individiiai
specificity of the myeloma globulins, more partienlnrly tbo'^e having tnobilif if-^ of
the same order as beta globulins. Later work by Slater. Wanl aiui Knnkel f ll'.'i.'.).

Globulin

(V ~

Globulin fi a Albuntr
1 J L_Ii t

normal'

I ' !‘

> — sUiJiO

■\v'

•

normal’

#■ ^

*R’

'normal’

’T’

— ~~*' -'*~

•

tiotnal

•

G’

—

C«J.

^normal’

•

'C'

. "

V V

♦

Fif 1 • anti - K

nimKvis of -om "W," “ I' " •• T “ •• f • - i

■til so I3,J* Pq fV|« *

,, The recent St “Th> gfebit,

-- •» <'-v

714

P. HOUGHTON AND N. H. MARTIN

globulin are concentrated in the same way, it is not difficult to collate the physico-
chemical differences with the immunological similarities in this complex group of
proteins.

The production of a “ biologically ” satisfactory protein takes place in three
phases, the assemblage of aminoacids and their linkage through the usual con-
densation of the terminal carboxyl of the one with the alpha amino with adjacent
member in the series, the total or partial coiling of the chain so formed into a helix
and the final folding of this helix ; the second and third phases being associated
Avith cross-bonding giAung S— -S— bridging, and other forms of cross-linkage of
a less Avell-defined and perhaps less stable character. The effect of this folding is
to determine the number of reactiAm groups on the outAvard surface of the molecule
and their relationship one to another. Specific antigen and antibody reactions
are determined by the “ pattern ” of these groups and their accessibility.

I 21 the present examinations the antibody reaction AAuth the globulins hi the
indiAudual myeloma sera Avas constant, AA'hether the antibody Avas produced to the
specific globulins of that patient, or to globulins from other patients, or to globulins
from normal people. It would seem then that, if this reaction is related to surface
“ pattenr ” on indiAudual molecules or groups of molecules, these patterns are
present in the globulins in normal patients as Avell as those suffering from myelo-
matosis, the essential difference being the frequency Aidth AA’hich one patteni
recurs, that is a quantitatiAm rather than a qualitatiAm difference.

In the hundred cases of myelomatosis -\ve have examined, Ave haA^e JieA'er
obtained any eAudence of a hereditary defect suggesting a genetic origin. Nor, so
far, have Ave any evidence of internal variations of aminoaeid sequences peculiar
to one or any of the myeloma proteins. Many of the demonstrated differences in
shape, in charge, and in predominance of a given immunological pattern, may be
explicable in terms of a defect of the final folding of the chain rather than in A^aria-
tions in the aminoaeid sequence similar to those demonstrated in the haemoglobins.
It is possible these changes depend on the ratio AAUthin the molecule of a helical
to non-helical configuration and this possibility is being tested.

SUMMARY

(1) Abnormal globulins of A^arying mobilities haA'^e beeii examined from fiA’e
patients afflicted by myelomatosis.

(2) The ultracentrifugal and immunochemical analyses suggest that these
globulins shoAA^ a progressiAm dcAuation from the normal rather than falling into
well defined groups. The cause of this deAuation is discussed.

Our thanlis are due to the British Empire Cancer Campaign aa'Iio defray'ed the
cost of this AAmrk.

REFERENCES

Bayne-Jones, S. and Wilson, D. W. — (1922) Johns Hopk. Hasp. Bull., 33, 119
Fkanglen, G. T., Martin, N. H. and Trehebne, J. D.— (1955) J.clin. Path., 8, 144.
Koenig, V. L. and Pederson, K. 0. — (1950) Arch. Biochem., 25, 97.

McConnell, R. J. and Martin, N. H. — (1958) Brit. J. Cancer, 12, 264.

Mcfarlane, a. a.— ( 1935) Biochem. J., 2d, 1176. ,r j oso

Muller-Eberhard, F. and Kunkel, H. G. — (1956) J. exp. J/erf., 104, -53.

MYl'.I.OMA c;i,OIiri,1NS

7ir.

Nevrath. H. ani) l?An.i;Y. K. — (MtAIt) * Tlu* \ ol. I, Scrlion [{. N’fw ^ nrk

(Acnclptiiir p. (mP.

Porter. R. R. — (1!'.")!)) Hinrhrm. 73. 1 l!t.

Proom. H. — (1P43) Pnih. li'trl.. 55. 4lP.

PcTX.AM, I'. W. — (IP.')()) ffll. rnwp. I’/n/'inl.. 47. 17, Siip]>l. i.

Idem AXP Unix, B. — (1P.')3) •/. hiid. ('hrm.. 202. 747.

Robixson. a. — (1027) Jhil. r.rp. Pnlh.. 8. 4.74.

ScHEiDiGOER. .1. 10.7.7) hil. Ari'h. AUfrijij. AM'.. 7. KEl.

.SuTER, R. J.. WaUD. .S. .M. AXI) Kt XKl;r,. H. (!. — (l!t.7,7) ./. r.r/l. Mnl., tot. .‘'.7.

Taa'Lor. .1. F. — (10.72) Arch. JSinrhrm. Htophi/^.. 3G. .'$.77.

W.AELExirs. G.. Tuai t.maxx. R.. KrxKr.i.. H. (1. AXn I'iiaxki.ix. M. ('■. (10.77) . 1 . hinl.

Chim.. 225. 2.73.

Wettz. B.— (10-72) -P Ilijy.. 50. 27ri.

BniRM.AXx. H.. Wfxnr.ut.Y. ('. L. axd Hassu:. A. — (10.70) Ih-it. ./. /-.rp. Path.. 31.
507.

716

CELL DIVISION IN AN ASCITES TIBIOUE IN VITRO

IViTH Especial Eeperekce to Abeoeihalities of
Cytoplasmic Cleavage

D. C. ROBERTS and D. J. TREVAN

Frcm) the Division of Experimental Biology and Virology,

Imperial Cancer Research Fund, Mill Hill, London, N.W.l

Received for publiefltion October I, 1960

The concept of stem lines within populations of tumour cells is nmy well
establislied, having first been put forward by Makino and Kano (1953) and by
Hauschka (1953) on the basis of experiments witli ascites tumours of rats and of
mice respectively. According to this concept there are within certain tumour-
cell populations one or more lines of stem cells which are capable of regular
mitosis and which are the primary contributors to the growth of the tumour,
Avhile outside the stem lines there is a variable proportion of genetically un-
balanced cells, the whole population being in a state of dynamic equilibrium
which may be altered by appropriate stimuli (Hauschka et al, 1956).

The purpose of this paper is to record the behaviour of cells from sublines of
the derived ascites form of a iiaturally occurring murine epitlielioma, at and
about the time of their division, and to draw attention to the lability of the
jirinority of cellsin the population whicli divide in an abnormal manner under the
conditions of our experiments.

jMaterials and methods

The tumour cell populations used in these studies are from two derived ascites
sublines of the tumour Epithelioma 255. Epithelioma 255 was found on the skin
of the flank of a 2I-year-old stock male C/57 mouse in 1955 ; it proved to be a
well differentiated squamous cell carcinoma, and since that time has been main-
tained bj' one of us (DT) in mice of the strain of origin.

The standard culture medium consists of 16 per cent calf serum in Hanks’
solution containing 0-07 per cent each of sodium bicarbonate and glucose and
0-35 per cent lactalbumin hj'drolysate (“ Bacto-Lactaltone ”, Difco) expressed
as percentages of the total volume of medium. No antibiotics are used. This
medium has a pH of 7 - 1 when in equilibrium with 5 per cent carbon dioxide.

Before preparing a population of tumour cells for examination, all apparatus,
instruments and solutions required are placed in a hot room at 36-5 G. aird
allowed to come to temperature. A mouse bearing an ascites sublinc of Epitheli-
oma 255 is killed, pinned to a cork board and immediately transferred to the hot
room in which all subsequent manipulations are done. The abdominal skiii is
reflected and OT ml. of ascites fluid is draum into a syringe already containing
0-9 ml. of culture medium. This is mixed and one drop is expelled into 1 ml. oi
culture medium. The resulting dilution (about 1 in 250) is mi.xed and drawn up
into a second syringe.

I'lvt.vioN IS vni:"

717

•\ of llii> irMiltiiur .MO-j'i'iO-io-.i ol liiinotii !•< int t. ■'lun-.l ml-, n

cuItuiT rlum)n-r {\\oWx\^ no.l Ttwao. \WO) ul.i.'l* i- pl'"'-''
of i\ ('iiu’niii'r('SO(']t(' in llio ^;uin’ liol tomu ( ln'\iiii inn! lioln'tl''.

I'.UU') or of a transjoirlaltlo t’ini'nni'to'-oo])i> (I’vloi'-. liol't-rl ainl liovaii. l.'o't)
also at '.tu ri C. \vlni'l\ is Uopt in a noiitlil'ontint: iainivatory. ■I'lto in tli<-

cliainl'prs an’ inaintainod I linnnrliont tlio ovinTiinonl in ri|niiilninin mill a
wator-satnratod atino'-plion' of .’i ^tor oi-nt oarlioii ilioviiio in air. and tlio cnllnro'-
aro foil at appmpriatt' inlorvais (mnally ovi’ry 1 !l days)- I’lia- c contra'iI oplio'i
arc' used on tin' cini’inicro^c'opo'- and tiinodap'-i' oinoplioloinicti'p*'*!’^*'' f cords
are taken conlinnonslv tlironidonit tlio oour.'o of l!i'' lAporiinont on !•> inin. s|on
]ian lU’gativo tiiin at tlu' rato ol ono Iraino every ‘Jl or ■It' s'a’onds,

Tlu' cinc’ n’conls an* analvsc'd in t!i<' tirst instance as neeatives (^^e develop
onr own lilin lent normally have ni'calivc’ tilm printi’d only when "e reipiin' to
asspinhlp. in oiu’ tihn. sccpu'iices from tin' ri'corils of a nnmher of evperinients).
a standard lO mm. jirojcctor with a 1 inch projector h'ns and a throw of I'J feet
being used. WV find that the aimle siihteniled to the eyi' is loo large for the
accurate examination of the whole lield of view on the screen at one time . I hi'
screen therefore' is divide'll into th’i sipian's of I'lpiid si/e and a lilm is .shown
either to a single ohservc'r for the rc'ipiisile nnmher of limes, or onei' to si'venil
observers who each critically c'xamine a part of the field.

A recent accpiisition is a Moray 1<» mm. fdm editor which, iisi'd in conjniK'lion
with a Premier footage and frame' connti'r. is proving invahiahle for the analysis
of complex sccpiences and indispcnsahle whc'n events have to he timed.

itKSfi.T.s

When (ir.st explantcd and viewed on the jihase mic'roscopc the cells apjiear as
spherical hodic.s with no disccrnihle interna! structure' siirroinnh'd hy a thin rind
of cytoplasm wliich at times shows undiilnting movements, l.ate'r in llie exjieri-
ment most of the cells llattcn on the glass, and much internal stniclure' I'lin then
be seen; nuclei, perinuclear granules and mitochondria r.re visible and the e'ylo-
plasm is bordered by an undulating membrane. In some circumstanc'cs thi' c'clls
orientate themselves one to another and make intereelhilar I'ontacts of a kind
never seen between cells in the rounded form.

When a cell divides in a " normal " manner it is as follows. ’Phe cell snddc'iilv
releases all firm contacts with the glass sub.strate and with any siirroimding cells
ancl the cytoplasm quickly shrinks until the cell ajijicars as a spherical hodv in
which little or no internal structure can be seen. .After a varying period of ('om-
plete immobility the spherical body elongates .slightly and an indentation appears
on either side which deepens until two apposed daughters are formed. 'Phe
cells now quite suddenly flatten out, the interna! structure can again he sei'ii
an undulating membrane reappears at the edge of the cytoplasm and once Inorc
the cells can move by their own efforts or make cohesions with their fellows
according to the conditions of the experiment. In cells dividing parallel to I lie
plane of the glass, it is common for the two daughter cells to spread out siniiil
taneously, although sometimes one expands before the other.

An expanded cell is usually attached much more firmk' tn fi,„ „i t .
th» arounded cell. In the event of a cell dividm7«rSt
glass, the daughter in contact ivith the glass completes a “ imimnl " d!vis"o,m

71S

D. C. KOBERTS AND D. J. TREVAN

that away from the glass, liowever, remains spherical with no discernible cyto-
plasmic movement, yet shifting as a whole in a quick and aimless manner as if
wafted about by the movements of the underlying expanded cells. Immediately
the spherical cell touches the glass, it flattens out.

Sometimes when a member of a sheet of cells rounds up to divide it is extruded
onto the upper surface of the sheet, apparently by the pressure of the cjdoplasmic
borders of the surrounding, firmly attached flattened cells. Again the rounded
daughter cells make no attempt to spread until they make contact with the glass
substrate. Thus we can determine a period during which the cytoplasmic
properties of an ascites cell differ from those of its interphase neighbours in an
identical environment. This we term the functional mitotic interval ; it is the
period between the rounding up of a cell before division and the spreading out of
a daughter cell after cleavage. The interval may conveniently be divided into
two parts ; the period from the rounding up of the cell to the beginning of cyto-
plasmic constriction and from that constriction to the spreading of a daughter
cell.

Fig. 1 shows a distribution graph of the functional mitotic intervals of the
243 cells in division that we have analysed ; we have excluded from this figure
cells that lie on top of the sheet as the time at which these expand appears to be
dependent on chance contact with unoccupied substrate rather than a function
of the physiological state of the cells. It will be seen that the functional mitotic
intervals of the cells that undergo “ normal ” division show a wide range of dura-
tion to produce a skew curve with a long tail in the high time range and a mode
at 51-60 minutes. Although the numbers are small, it appears that the functional
mitotic intenmls of the cells that have been seen to divide abnormally probMy
follow the same pattern as those of the cells that divide “ normally ”.

ABNORMALITIES OF CVTOPLASMIC CLEAVAGE

Fig. 2 indicates diagrammatically the abnormalities of division which we have
seen, and roman numerals in the text refer to this figure.

The most simple abnormality of cleavage recognised was the persistence of a
thread of cytoplasm joining two daughter cells when they spread out after the
division of a uninucleate ceU ; this tliread parted to form two free daughter
cells {II). In this series such cjd oplasmic threads were seen on 8 occasions, and the
times from the spreading out of a daughter cell to the breaking of the cyto-
plasmic thread varied between 18 and 43 minutes except in one cell when the

thread remained for 138 minutes. j ,• i

Sometimes a cytoplasmic thread failed to break ; and instead thickened
until the two daughter cells came together to form a binucleate cell (III).
we have seen on 7 occasions, the time between the spreading of a daughter and
the formation of a recognisable binucleate cell varying between 42 and 3
minutes. We were unable to foresee from the appearance of a thread whether it
would break -with the formation of two separate daughters or persist with t e
formation of a single binucleate cell. A similiar process was seen m 7 cells in
which there was either an abortive attempt or apparently no attempt at cyto-
plasmic cleavage, and the cell, which was uninucleate when it rounded up, was
seen to be binucleate when it re-expanded (IV).

720

D. C. ROBERTS AND D. J. TREVAN

Another mode of formation of a binucleate from a uninucleate cell was seen
once only. A uninucleate cell rounded up to divide and cjdoplasmic cleavage
began 23 minutes later. Nineteen minutes after this the first daughter cell

0 -

©-©

-1

0 -

©KD

©:

III

- (• •)

(. .)-

0©

(. .)-

0©

VII

(. .)-

(. .)

VIII

(• •)-

(T-l)

•

- (• •)

•

•J ~

'*• •

.* v

- (• •)

XII

Fig. 2.- — ^Diagram of abnormalities of cytoplasmic cleavage seen in ascites cells of Epithelioma 255.

spread out on the glass to be followed by the second 14 minutes later. The tuo
daughters appeared to be entirely separate from one another and despite re
peated examination of the cine record no connecting thread has been seen. er
a further 554 minutes however, the two cells came into close apposition and leir

CELL DIVISION /A’ VlftW

cytoptam fused lo form a |;ni™>li 'm'^! iu!lr.',l "o'

nLLrily eomm Ued to ' f J™ ' ''i"”'-'™"- T

Im-e not seen a binnclcatc c 1 ■ ‘ J , i,i„„ck’!ilo colls round \\\> to divide,

division. On S-— 0> donpl,...,.

cells (VI). On only
division

S^ETuarnearirconti'i"®-'^ •Om

to be ioined bv a cytoplasmic thread which disappeared nflm Sd mi

iiiiiiiites and ii

to be ioined by a cytoplasmic - , . i- ,i „,,ii

single binneleate cell was again formed (Vll). In the seeo.nl ease a bimieleate -e
rounded np, C2 minutes later an abortive attemiil. at cleavage- was seen ami it
was not until a further 4i)0 minutes bad clap.-^cd that the cell spread out auaiii.
still containing two nuclei (VIH). The thml cell was seen t o divnh- several t iim-.K.

and will be considered later. _ i • i

Only once have we seen the formation of one binnolenle and one niuiniele.ile
cell from the division of a single binnclcatc cell. 1 bis cell ronmh*d nji and alii-r
75 minutes underwent a 3-way division. After n further -la iiiiiinles. two of tin-
daughter cells expanded to he followed by the third after a fnrtlu-r (iP miiinl<-s.
This third cell was seen to be joined to one of the other daughter cells Uy n thin
cytoplasmic thread which persisted for 4;) inimile.s : it was flien aiisorln-d and a
binucleate daughter was formed (IX).

Successive divisions each with failure of cylo])lasmie cleavage givt- rise- to
very- big cells with largo and complex nuclear sy.stcms. nlthoiigli this may In-
followed by the death of the cell. Thus we liavc seen a niiimu-Ieate (-(-II whii-li
rounded up and cytoplasmic cleavage began H minutes later : eleavagi- was not
complete and 140 minutes afterwards the ceil spread out again in a iiiinieli-ato
state. After a further 1062 minutes the cell rounded iiji again and an (-arlv
cytoplasmic furrow rvas seen after 45 minutes. Sixty-six miinitcs lati-r the ceil
attempted to divide, failed, remained as a single cell and after luiotlicr 2,s.5 mhintes
expanded to reveal 4 nuclei. It appeared to be cpiitc healt hy. Imi after a fnrtlu-r
231 minutes the cytoplasm suddenly coagulated and it died '(X).

Another cell contained two nuclei when fir-st seen. It. rounded up for tlie
rs time and a cytoplasmic furrow rvas visible 5J minutes later. Cvloplnsmie

further 38 minntc.s it expanded m'aiii
up P28 minutes and (hen roiimh‘.i

Sm” an atSn,-r;p . -“J™;

nuclei (XI). spread out once more, .still witli .(

ce l which rounded up to dinVlP^ fntinu 4 - ' •‘’’‘^en a

uninucleate

bim,r.u„: . rounded up to divide failorl i , '

''''''8kters(Sl)."'^’''' s^^cc.s.sfully Tniriwo mdnuclette

722

D. C. ROBERTS AND D. J. TREVAN

DISCtrsSION

• preparations used in this study were prepared in such a iray as to

give the best possible visualisation of the cytoplasm. The refractive index of
the culture medium was too low to permit analysis of the behaviour of the
chromosomes during cell division, and we do not propose to discuss here the
eiiects on ploidy that may result from these aberrant cell divisions. The
examination of cells in a high protein medium as described by Barer and Joseph
(1955) might well allow the nuclear behaviour of similar cells to be studied and
correlations determined. Comparisons could then be made with the modes of
formation of binucleate and polyploid cells in non-neoplastic tissues as described
in the regenerating liver of the rat by Beams and King (1942) and in cultures of
mouse fibroblasts by Fell and Hughes (1949).

The picture that emerges from this in vitro study is that of a population of
cells in which the great majority of cells that divide do so in an apparently normal
manner while a minority show a wide range of abnormalities of cytoplasmic
cleavage. The cells resulting from these abnormal divisions are usually viable
and have frequently been seen to undergo further division which may appear to

EXPLANATION OF PLATES

Fig. 3-5. — Formation of a binucleate cell from a uninucleate cell.

Fig. 3. — A uninucleate cell (A) before division, x 280.

Fig. 4. — Daughter cells joined by a cytoplasmic thread, x 280.

Fig. 6. — The thread has been absorbed ivith the formation of a single binucleate cell, x 280.
Fig. 6-7. — Formation of a binucleate cell from a tminucleate cell.

Fig. 6. — A uninucleate cell (B) before division, x 280.

Fig. 7. — Resulting binucleate cell from (B). X 280.

Fig. 8-11. — Formation of a binucleate cell from a uninucleate cell.

Fig. 8. — Uninucleate cell (C) before division. X 330.

Fig. 9. — ^Tivo apparently separate daughters (D) and (E) from the division of (C). x 330.
Fig. 10.- — Fusion taking place between (D) and (E). x 330.

Fig. 11. — A binucleate cell (G) resulting from the fusion of (D) and (E). x 330.

Fig. 12-15. — A uninucleate cell which divides to form a single binucleate cell. This cell then
divides to form two uninucleate daughters.

Fig. 12. — ^Uninucleate cell (H) before division, x 330.

Fig. 13. — Binucleate cell (I) resulting from division of (H). x 330.

Fig. 14. — Binucleate cell (I) just before its division, x 330.

Fig. 15. — Two iminuoleate cells (3) and (K) resulting from the division of (I), x 330.

Fig. 16-19. — Formation of one uninucleate and one binucleate daughter from the division of a
single binucleate cell.

Fig. 16. — Binucleate cell (L) before division. X 280.

Fig. 17. — Three-waj^ division of cell (L). x 280. ,

Pig. 18. Daughters from division of cell (L). Two daughters are expanded while one is still

rounded up. X 280. .... r it \

Fig. 19. — Binucleate cell (M) and uninucleate cell (N) formed from the division ot coll (l>).

X 280.

Fig. 20-23. ^Formation of cell rvith complex nuclear structure from a binucleate ceil.

Fig. 20. — Binucleate cell before division, x 330. .

Pig, 21.- The cell has attempted division, there has been failure of cytoplasmic cleavage, ana

a binucleate cell is again formed, x 330. . j „

Pig 22 Attempted division has again taken place; cleavage has again faii^ anu a smgio

4-nucleate oeU has been formed. Only three of the nuclei are visible m this tipiro. X JJV.

Pig, 23. ^The 4-nuoleate cell has attempted division, cleavage has again tailed, nitn tiie

formation again of a cell with 4 nuclei, x 330.

Buitish .Toup.nai. or Canckk.

Vol. MV. No. I.

^‘Obori.s iind T

Br.TTISH .JoUIlN'AL ov C'ANcnit,

\’ol. MW N... I

V..I MV. N" <

Br.iTisii .TornSAi. or CANrKi’..

CELL DIVISION IN VlTIiO

a “rma, be bLgU abo.t by a selcetivc proeeae achnj; '

cell population wherebj' a variant cell lino is raised niiinencall\ n an
critiLlVoportion of the population. This variant line can then displaro t m n-
petitive cell lines and become manifest by changing tlic clinractcnstins of tin

We do not know what environmental or other factors delerinnu' llm run-
tent of the minority cell population that wc have described, nor wlietber Uu'
changes that we have obsenmd in vitro reflect similiar bcliaviimr in riro nr
represent potentialities of the cells that remain unfulfilled in the absence of .special
factors in the animal host. It is possible, however, that this changing minority
population may represent a mechanism whereby variant cells may arise, wliicb.
if environmental factors are such as to give them a selective advantage over tlu’
rest, may be the progenitors of a new stem cell line.

SUMMARY

This paper records the behaviour in vitro of cells of an ascites suliline of n
naturally occurring murine epitheUoma at and about the time of their division.
Although the majority of cells divided in what appeared to be a normal manner,
abnormalities of cleavage in a minority resulted in a minority poimlation of
vmant ceUs the content of which might alter from generation to gcnenition

JJlXiftecu'Sf'*”''

We wish to record our indebtedness to Dr. H. B Fell F T’ S l.nti, r i
encouragement of our work and for her help in the presStlotVtIm rc.^^^

references

rr,.,i„ H. B, a™ HuXs a rl S fl’"!; ’f- “i-

Havschka, T. S—dgsaw 'micr. Nci., 90, . 3,^5

H», KvmABTB, T Gmi'- ST'

63, 083. " *'■- A».08, D. ,

j>y A.-;, ToBVA^af » (i" P»S8).

724

SHEET FOEi\IATION BY CELLS OF AN ASCITES TUMOUR

IN VITRO

D. J. TREVAjST A^’D D. C. ROBERTS

From the Division of Experimental Biology and Virology,

Imperial Cancer Research Fund, London, K. jr.7

Received for publicntion October 1, 1960

Ix the course of a stud}'^ of tlie behaviour in vitro of cells from an ascites
epithelioma of the mouse, it was noticed that the cells could aggregate into sheets
of varying stabilit}'. This paper describes the phenomenon and some of the
factors b3'- which it is influenced.

JUTERULS AXD METHODS

The cells to be described are from one of several ascites lines derived bj'
deliberate selection from a murine epithelioma. This tumour arose on the back
of an untreated male G/o7 black mouse j^ears old. It has been maintained
since 1955 by serial grafting in mice of that strain to which it is specific. It is
designated “ Epithelioma 255 ” : the details of its changing histolog}’ following
different selective grafting procedures will be described elsewhere bj’ one of us
(DT). The ascites line concerned in this account is designated " Bl ”. All
ascites lines are maintained by serial intraperitoneal passage of peritoneal exudate
diluted in phosphate saline to a degree depending on a rough visual estimate
of cellularity, iisuallj' between I in 3 and 1 in 20.

Mice bearing the ascites line Bl show evidence of peritoneal exudate at from
10 to 14 dajfs after inoculation. Thej’^ hai e been killed to j'ield cells for stud.y at
times after inoculation ranging from 10 to 38 daj’s, but usuaOj' at about 18 da,vs.
Tj^picalty at this time there is one to 3 ml. of exudate containing about 10’ cells
per cu. mm. and an amount of blood estimated visuallj’ at between 5 and 25 per
cent of the volume. Solid tumour is present as a craggj’’ mass about 20 X 5 mm.
in the lesser omentum and around the pancreas, as multiple nodules in the
mesenterj'^ and as a thin sheet on the parietal peritoneum. There are multiple
minute secondarj’’ deposits and haemorrhages in the lungs. iMice killed earlier
show less ascites often with less blood and less solid growth ; at 10 or 14 da.vs
there are usualty haemorrhages in the lungs but onl}’ microscopic metastases.
Mice surviving be^mnd about 18 daj’s often accumulate up to 7 or 8 ml. of ascites
and much more solid growth, while the lungs are grey and rigidly consolidated
with secondarj'^ tumour. Lung haemorrhages tend to be less severe in long
sumdvors.

Cells for observation are taken from a mouse killed with coal gas a few minutes
previously and kept warm, bj’ aspiration into a pre-warmed siliconed syringe ot
medium. A secondarj’ dilution to a final degree of about 1 in 250 is made in the
same medium, which "consists of Hanks’ saline Avith 16 per cent of calf serum.

CELL SHEETS IX Vimn

0-3o per cent of lactalbnmiu hydrolysate (Baclo-Iactallone. Difro). and n'(i 7 |,vr
cent each of sodium bicarbonate and glucose, ail cxjtrcsscd ns inTt'cnlniic- of tlm
whole medium. The pH of the medium is 7-1 when (•(piilibrnlf'd wilh T. p.T rent
carbon dioxide in air at 37° C. The cell suspension is (lien injeeted into n elinmber
(Roberts and Trevan, 1960a) through whicli 5 per rent carbon dioxide in air
passes, and the cells resting on the lower glass sliji arc c.xnniined with an invert' d
phase-contoast microscope at 37° C. and photographed by time-lapse rinenial'e

adjusted to Jiroduce a linal eoneentr.it i-.n of
cells of between 500 and 1000 per cu. mm., which in (liis elianiber. in a drop

15 ram. deep, gives about 5 to 10 cells in the field of a X2(i objective at the Mart
of an expenment. 'j' ' i oi ai me Mart

-lls vary- ' »

raffled border, others a™ ne.v f ' “"■““'“I'll kv ncliv,.lv

raffled border separated by sMoth mo'’" “1 ^ rviti, .aroi or"''

•reas form p.seudopodii and a "elf t™? rap o '' n . “ m''''

' WfXdia or :

=es Sfet^f LoffhT"/ 'f "T -

'Vitbdratvs followkT; tt >l«ng te l/„e rf " ''f ralf

*epy eoneave ndthtoe rfffl"d a""?" "‘'‘I’' '>«»■• eell rnff”' " "»■>'
In Other cases first the otJier ce I

alternately become inacthS i ^ cell anTiL ! ooneavit v

toacal cavitv ^ nientioned tL,aT ' ^”3' I"me in fim it .

glas.s they separate "Hu fresh ^fr '‘"^Inre.

f^troiJy tn ^P^’ead out Inf ^rst attach i ^ ‘'"t- uon.

'» hio.b’e
"it /lout

726

D. J. TREVAN AED D. C. ROBERTS

If, however, the culture medium is not renewed, the cells in such aggregates
form increasingly lasting attachments to each other during the next 12 to 30
hours, so that a culture between 40 and 60 hours old in which there has been no
change of medium contains many areas in which cells that are still flattened on
the glass now cohere, still as a monoIa 3 rer, resembling normal epithelium. Here
and there among the cells can sometimes be seen fine intercellular strands like
those between normal prickle cells. Only the cells at the edge of the pavement
still shov' active movements of their free borders, and there is a general diminu-
tion of motility, in spite of which a small sheet may move across the glass as a
whole in a manner that demonstrates the firmness of the attachments between
its members. A sheet increases in extent by the attachment of more cells
migrating to it on the glass, and bj’’ cell division, which continues for at least 15
hours after the formation of such fixed sheets. If a change of medium is still
withheld, the activity at the edges of the "sheet gradually lessens until none can
be seen. Ultimatelj’’ the cells shrink, fall apart and die. A minority of the
cells in the population never adliere to a fixed sheet when thej'^ meet it ; maii}'^ of
these are the giant, often multinuclear, cells produced by aberrations of division
(Roberts and Trevan, 19606). We have a film sequence that shows a stable
sheet being formed as a result of several successive divisions in an initialty small
group of cells in contact. The daughter cells from each division adhered firmty
and promptly to their neighbours in the sheet ; nearbj^ were several larger ver}'
actively amoeboid tumour cells that adliered neither to each other nor to the
growing sheet.

If the culture fluid is removed and replaced with fresh medium after the
formation of fixed cell-sheets and before the death of the culture there is an
immediate renewal of amoeboid activity and the adhesions between cells in the
slieet break. The cells form a sliding sheet or migrate apart ; if division has
ceased it begins again. As time goes on, however, adhesions agaui form between
cells and unite them in stable sheets. This happens more rapidly than it did in
the fresh culture, i.e., in about 10 as against 30 hours ; the culture is now more
crowded than it was when fresh, and the more crowded a fresh culture is the more
quickty stable sheets are formed. A stable sheet re-formed after feeding the
culture usually breaks up again at the next feed, but less completely, and it
re-forms even more quicklj'^ than before. If the medium is renewed ever}"^ dajq
stable sheet formation maj>' be postponed at least until the point when the culture
becomes so crowded that many cells die in spite of the frequent changes of medium.
Cells in contact remain in sliding sheets throughout.

On three occasions stable sheets have been subjected to the replacement o
the culture fluid with medium from which the glucose has been omitted. Ihe
sheets remained stable and adhesions between the cells did not break.

Discussiojr

When the cells of this ascites tumour derived from a well differentiated
murine epithelioma are maintained in culture on a glass surface in a goo s a e o
nutrition they flatten, adhere to the glass, and migrate upon it. M hen tliey mee
they slide around but not over one another, remaining in a
do not cohere. When pseudopodia of two cells meet, one of the pseudopod
may inhibit the activity of the other, as though it were ’ dominant . Such

CELL SHEETS /-V V/T/fO

ll'il

till-

tenance of a monolayer . contic ■ vLercroinltio Hcavniimii ntnl

cell cohesion, and is believed celfs S ->'■

Kart.lm»ser ( 1957 ) later reported X„ „,l,er a.-.rrn,„a

..lay be rcEartW a, i,..c.r„...,lia... bet...™ . »-
two extremes; they inhibit one another's movements more limn i-'^reonm ■11
a t ' . „'4. TLio Tpiiiiniseent of tlie obsorvalums of nml

made cinematographic records of the re-aggi

what part if any was plaj’ed by specific ava...v,o.„

Weiss concluded that “ Cells in homologous, as well ns heterologous, groups l;i‘<>p
shifting about one another in a maimer which rules out stable mutual attach-
ments as a primarj' event in aggregation. The only differouce between the
homologous and heterologous groups seems to be that homologous ei'll.s lend to
retain mutual contact along a broad front whereas heterologous eomhiiiatioiiv
tend to break up soon after contact He illu.strates the break-up of a heti-ro-
logons combination by a photograph of a pseudopodium of a liver c(>ll within a
smooth concavity on the surface of a lung cell : this strongly rc.sembles what may
happen when two cells of Epithelioma 2oo meet; on the other hautl. the'V
epithelioma cells also form aggregates, which we have called " sliding .«h(‘cts ",
which resemble the aggregates of homologous epithelium seen by Wei-^s :uni
Taylor, iu that “ they retain contact along a broad front " without coheriiiL'.

• pseudopodium over another might be oxidaiued

m he following way A cell puts out a pseudopodium bv virtue of it.s Lnfaet
nith, and adherence to, the substrate. The advancing ed-es of onno-ed nv^c.Kh.
podia are thin and sharp ; when they meet either edge may unde cut 1 > r
and loosen it from the substrate so that it retracts cfn^Hn^u '
from the edge inwards. Further advance of tbe progrc.s.-ively

detaches more of the other from the substrate • if thhi"n
cut cell margin has an opportunitrrSr V V

other cell in turn. It may be a^mattJr nf^ again and may undercut the
undercuts the other. Once a cell border which p.seiidoiiodiiirn fir.st

likely to „„derc„ fc lert | 'L"'!o™h'': »"<' b

728

D. J. TREVAN AND D. C. ROBERTS

gradual lessening of niigrator}'^ activity that in the end accompanies relative
starvation. This behaviour is analagous to contact inhibition. Abercrombie,
Curtis and Karthauser (1958) have reported that some failure of contact inhibition
may be produced in cultures of fibroblasts by a high concentration of embrjm
extract. Our observations of Epithelioma 255 also suggest a connection between
nutrition and contact behaviour. There is no embrjm extract in our medium,
but the administration of medium containing little or no glucose to a stable
sheet of cells of Epithelioma 255 does not cause it to break up ; this may indicate
that the formation of intercellular adJiesions is due to a reduction, by consump-
tion, of the glucose available. Alternatively the cohesiveness ma}’’ be induced
by the consumption of some other nutrient or the production of some metabolite,
and possibly the cells fail to part companj'^ when fed with medium lacking glucose
because they have not enough energy to migrate apart ; the effect of glucose-free
medium on cells in the earl 3 % activelj'^ migrating cohesive state has not yet been
tried.

Dabrowska-Piaskowska (1959) appl 3 'ing Moscona’s techniques to tumours
found that cells of a murine mammar 3 ^ tumour reduced to a single-cell suspension
vdth tr 3 ^psin would re-aggregate into a structure resembling that of the tumour
in vivo, when cultivated on glass or on a plasma clot. The sheets formed by
Epithelioma 255 cells might be considered as analogous attempts at reconstruction,
indeed, the 3 ’' show intercellular bridges M'liich give them an at least superficial
resemblance to prickle cells. The dependence upon nutrition in vitro of this
apparent differentiation of the ascites form of Epithelioma 255 is of interest in
that further stud 3 ' of the factors involved ma 3 ’^ throw' some light on the mecha-
nisms w'hereb 3 ' the cells of a malignant tumour in vivo ma 3 ' var 3 ' in invasiveness
or remain dormant for long periods.

SUMMARY

Cells of a murine ascites epithelioma w'hen cultured on a glass surface in a
medium consisting of calf serum, lactalbumin hydrolysate, salts, bicarbona e,
and glucose adhere to the glass and move about on it. When tw'O cells mee on^
may inhibit the pseudopodial activity of the other, but the 3 ' do not cohere a is
stage ; at a later stage, if the culture medium is not renew'ed, adhesions are
formed betw'een cells w'hich eventuall 3 '^ unite them into stable sheets resem mg
normal epithelium. Cells in these sheets may be connected by interce u
bridges like those between prickle cells. The sheets grow by accretion an
division of the cells in them. This behaviour is likened to the recons rue
normal tissues and of tumours from dissociated cells in culture tha as
observed by other workers. j.|jg

The formation of stable sheets may be prevented or reversed by
culture medium, and there is a little evidence that the medium ®
glucose in order to do this. The dependence of apparent differen la ^

environmental factors, possibly nutritional, justifies further study in an ^
to elucidate some of the mechanisms responsible for the variation in im a
of tumours in vivo.

We are very grateful to Dr. Honor Fell, F.R.S. for her interest in this
and for her help with its presentation.

CELL SHEETS IN VITRO

72!)

REFERENCES

Abercrombie, M., Curtis, A. S. and Karthauser, H. M. — (l!)o8) licp. Bril. Bmp.
Cancer Campgn, 36, 507.

Idem AND Heaysman, Joan E. M. — (1954) Exp. Cell, lies., 6, 293.
lidem and Karthauser, H. M. — (1957) Ibid., 13, 276.

Dabrowska-Piaskowska, ICrystyna. — (1959) Ibid., 16, 315.

Moscona, a. — (1957) Proc. nat. Acad. Sci., Wash. 43, 184.

Roberts, D. C. and Trevan, D. J.— (19G0o) .7. R. micr. .S'oc., 79, 301.— (lOfiOi) Bril.
J. Cancer, 14, 716.

Weiss, P. — (1958) “ International Review of CyAoIogy, vii, 391 ; Ed. Bourne and
Danielli, New York (Academic Press).

730

FATE OF 35S-CYSTINE IN JIULTIPLE SIYELOMA

E. SIEGEL, B. A. SACHS and F. A. GRAIG

From the Medical Physics Laboratory, Mediatl Division,

Montefiore Hospital, New York 67, N.Y., U.S.A.

Received for jniblicnfion October 13, 1060

The abnormal protein-bound polysaccharide associated with multiple myeloma
(Sachs, Cad}' and Ross, 1054) and the liandling of radiosulfate by patients with
this disorder (Siegel, Sachs and Graig, 1059) have been reported previously. The
present study describes the fate of ®“S labelled C3'stine in myeloma and other
malignant neoplasms, and compares these results with those published earlier
for radiosulfate.

JIATERIALS AND METHODS

®“S as 1-C3'stine (specific activity' of about 30 mc/g.) in doses of approximately
0*5 me was administered intravenously' to 5 patients with multiple myeloma and
to 5 patients with other neoplastic diseases (Table I). The assays of radioactivity
were performed with a gas flow counter, since is a pure, weak beta emitter
(0'168 mev maximum energy' ; 87 days half-life). Appropriate corrections for
self-absorption and back-scattering rvere made for the specimen geometry em-
ploy'ed as well as for phy'sical decay'. The detectors used were calibrated against
standard solutions furnished by' the National Bureau of Standards. Blood
was collected by venipuncture at intervals of 5 minutes, 1 hour, 4 hours, 8 hours,
24 hours, 2 day's, 3 day's, 5 day's, 7 day's, and 14 days following administration of
the tagged cystine. Blood specimens (about 15 ml.) were placed in test tubes
containing two drops of versene to prevent coagulation. Urine was collected for
the first 8 hours, the next 16 hours, and daily' thereafter for two weeks after
injection of cy'stine. ,

Calculations. — The concentration (per cent dose/liter) in -whole blood an

plasma (Fig. 1) and the fraetion of the dose excreted (per cent dose excre e )
in urine during 14 days following administration of the radioisotope were com
puted (Table I and Fig. 2). The apparent space of dilution was calculated roni
these blood and plasma data. This computation involved finding the hy'pot
coircentration at zero-time by means of graphical extrapolation in order to o ai
the so-called zero-time radiosulfate space (Ry'an et al., 1956). The ratios o
to body Aveight and body' surface Avere determined. Body' surface of eac i su J
Avas obtained from the patient’s weight and height using the conventiona u
relationship.

RESULTS

blood levels. — in Avhole blood, as Avell as in plasma, falls
first 8 hours and then declines sloAvly (Fig. 1). This pattern is foun m °
tiple myeloma patients and the other subjects. Since the leA'els an pa

35S-CYSTIKE IK MULTIVUi MYEEOMA

7:?i

-vhoip blofKl

aw f jt^tut^iosc/uToiTo vj"

Lr for to mth conccMlralion. have c roj.po,!

per cent dose/liter for the others. “ Us. Tliesc levels deerease

to about one-third of their g of tlii 8 hour vnlncs ai. Ti days after

slowly thereafter, being about three-quarters
the administration of the tagged cystii .

Fi(i. 1. — ^Thc variation of the mean ’*S whole blood concentration with time for five i)ntients
with multiple myeloma and five patients with other malignant neoplasms. Tlio range
(maximum and minimum values) is indicated by vertical lines extending from the individnal
points.

By graphing the logarithm of whole blood and plasma concentration
against time, it was found that the cunms thus obtained could be resolved into
exponential components. The curves from three of five multiple mj^eloma patients
have two decay rates. An initial rapid decrease, corresponding to a half-disappear-
ance time (Ti) of about 0-45 hour characterizes the fall of during the first
liour. I'he decline thereafter is quite slow, described by a half-disappearance time
n a) of about 280 hours (Fig. 2). An intermediate half-time (T^) of about 2-5 liours
however, was present for the other Wo myeloma patients during the I to 8 hour
mtovval following the injection of the doses. For the other group of patients
t hree distinct rates governed the disappearance of the (Fig 3 ) An initial Imlf
tune ( 1 ,) of about 0-26 hour for the first hour is followed for the iieS Tb x
an intermediate component (T,) of about 1-4 bourn Tr the .ne '"T

gradual fall is described by a half-time (T 3 ) of about 160 hours, 'aieanvldts tf

732

E. SIE&EL, B. A. SACHS AND F. A. GBAIG

these half-disappearance times for both groups of patients, as derived from tlieir
respective whole blood and plasma curves, based on the data collected over a
two week period, are listed in Table II. The figures reproduced here (Eig. 1 and 2)
cover oifiy the initial 24 hours following the administration of the labeled ammo
acid to illustrate more clearly the early phases.

Fm. 2. — Resolution of the whole blood ’’S concentration curve obtained from a patient with
multiple myeloma into two exponential components. T, and Tj are the respective initial
and late half-disappearance times. The various half-disappearance times given in the figures
and Table II are actually based on graphs covering the two week period of observation ; for
illustrative purposes, only the early values are presented here.

'S-CYSTINE IN* MULTIPLE MYELOMA

i:v.\

urinary excretion. — ^The excretion in the urine is relatively slow and

about the same for both groups of patients (Table I). The output of tl'ic first day
ranges from about o per cent of the dose to a maximum of about 20 per ccnl. of
the dose. From the second day on, the is eliminated in the urine exponent iallv
the average doubling-time for cumulative excretion being about 7-2 days (Fig. 4)
By the end of the first week, about 30 per cent of the dose has been excreted.

^'‘S-cystine spaces of dilution. — The zero-time radiosulfate whole blood spaces
and their respective ratios to body surface and to weight are given in Table J.
These results are similar for both the mj'eloma patients and those having other
neoplasms. The average space for patients with myeloma is 7-30 -b 1-41 liters
corresponding to 13-2 ± 3-49 x 10-’- liter per kilogram of body weight. For the

other patients, an average space of 8-66 ' -9 r-

rj,. . discussion-

734

E. SIEGEL, B. A. SACHS AND E. A. GRAIG

o » ir: C'l ^

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^0^0+ CH-

* Standard deviation.

®®S-CYSTIKE IN Min/riVLE MVEl.OMA

7:5.')

lasting a few days ; (c) the exponential portion of the curve. pre.Miined to u
primarily the rate of metabolic dcgraclation of the labeled jn-otein and (d . f
the experiment continnes for sufficient time, a phase retlect mg t he re-meorpoi at mn

of the isotope into the protein. r v ir *

In comparing the fate of tagged eystine with that of radiosulfale. .‘> 0.11 e .Mg-
nificant differences are observed. While the siiacc.s of dilution for lese no mi >-
stances are quite similar and in accord with those reported for radio.sultate lor
normal subjects as well as for others liaving nialignnticics (Siogci ct o .* • •> .

Ryan el at, 1956 ; Walser, Seldiu and Grolhnan, lUfiS), tiie handling and turn-
over of these compounds are quite different. Fig. 1 illustrntcs the ^ ’8 whole hhiod
concentration as a function of time for those receiving labeled cystine, hollowing
administration of labeled cystine the blood concentration decreases slowly ;
thus, by the second day, when the concentration following radiosulfato, as
previously reported by us, is less than 0-5 per cent dose /liter, the ^ 'S concent rat ion
from cystine is about six times greater. This markedly slow fall in blond levels
is characterized by the greatly prolonged late (T^) lialf-disappcarancc times
following labeled cystine (Table II) as compared to those found for radiosnlfatc.

Table II. — Initial, Intermediate, and Late Half-dis(tj)pe(irni)ce Timr.'i
obtained from Whole Blood and Plasma Concentration Carves.

Average half-disnppcnrnnee times (hours)

( V

Patients T, T- T,

Multiple myeloma . 0-448 + 0- 1.57* — t 281 ^ 77

Other neoplasms . 0-265 ±0-13.5 1-41 i;0-65 102 ^121

* Standard deviation.

t Tj -was found in two of the five multiple myeloma patients, nver-
aging 2 • 47 hours.

Thus, the average value Tg for patients with myeloma given ^'’S-cystine is 281
hours as compared to 15-9 hours follorving tagged sulfate. A similar lengthening
01 this paranieter is observed also for the patients with other neoplasms. There is
a corresponding lower output of in the urine from 35S-c3rstine as compared to
at lo lowing administration of radiosulfate (Fig. 4). By the second day when
ore than 60 per cent of the injected sulfate has been excreted, those given

triSn '^ose. Furthermore, for those

lative uSl"^ t '''' equilibrium is attained after the second day, the cumu-
noted output growing exponentially, with an average doubling-time as

736

E. SIEGEL, B. A. SACHS AND E. A. GRAIG

as well as the abnormal myeloma globulins. The present investigation did not
involve study of homologous serum proteins, and the curves obtained represent
summation effects for all the serum protein complexes. It is possible that if
individual globulins were studied, greater differences might have been observed
between the turnover of the ^sS-cystine labeled normal globulins and the labeled
myeloma globulins (Putnam, 1959).

There is a suggestive finding of a difference in the fate of ®“S-cystine for
myeloma patients as compared to those having other malignancies. The initial
(Tj) and late (Tg) half-disappearance times are longer for those having myeloma
(Table II). This suggests that the hyperglobulinemia of myeloma may result from
a slower catabolism of myeloma globulin. In this connection, Putnam and Hardy
(1955) observed a greater half-disappearance time for the turnover of beta mye-
loma globulin than that obtained for the normal beta globulins by London (1950),
and stated that the hyperglobulinemia of myeloma might be due to accumulation
of the myeloma globulin by virtue of its slow turnover rate.

SUMMARY

®®S-l-cystine (specific activity 30 mc/g.) in doses of approximately 0-5 me was
administered intravenously to five patients with multiple myeloma and to five
subjects with other neoplastic diseases. For both groups of patients, the con-
centration of whole blood and plasma were similar. The blood levels declined
slowly ; at five days after administration of the dose they were approximately
three-quarters of the initial 8 hour values. The mean zero-time spaces of dilution
were 7-39 ± 1'41 liters for the patients with multiple myeloma and 8*66 d; 2'67
liters for those with other neoplasms. urinary excretion was low, 13'6 to 30-9
per cent of the dose appearing in the urine within 72 hours. The turnover and
excretion of administered as cystine are very much slower than when given
as radiosulfate. By graphical analysis, it was possible to resolve the blood con-
centration curves into three exponential components. Patients with multiple
myeloma were found to have longer initial and late half-disappearance times
when compared to the subjects with other neoplastic diseases.

The authors wish to acknowledge the excellent technical assistance rendered
by Messrs. Joseph V. Marino, Jack Sokol, and David Weinstein. This investiga-
tion was supported in part by Grant No. C-3438 from the National Cancer Insti-
tute of the National Institutes of Health, U.S. Public Health Service.

REFERENCES

Abmsteong, S. H., Jr., Beonsky, D. and Heeshman, J. — (1955) J. Lab. dm. Med.,

46, 857. ^ r a ■ rrn or

Goldsworthy, P. D. and Volaviles, W. — (1957) A7m. N.Y. Acad, 70, ..b.
London, I. M.— (1950) The Robert Gould Research Foundation, Inc., Cincinnati, Sym-
posium on Nutrition, 2, 72.

Putnam, F. W.— (1950) Engl. J. Med., 261, 902.

/dem and Hardy, S. — (1955) J. biol. Ghem., 212, 371. r r r

Ryan, R. J., Pascal, L. R., Inoye, T. and Bernstein, L.— (1956) J. dm. Imesl.,

Sachs, B.’ A., Cady, P. and Ross, G.— (1954) Amer. J. Med., 17, 662

Siegel, E., Sachs, B. A. and Geaig, F. A.-(1959) Proc. Soc exp Biol., A . F., 101, o3.

Walser, M., Seldin, D. W. and Geollman, A.— (1953) J. dm. Invest., 32, 2J9.

IN THE LIVEHE

iCCMTOATroSOTMOSP™

W. J. P. ^?EISH AKD AlsTT RYLEI’l’

From the Cancer Research Unit, The University, Sheffield, 10

Received for publication October 1:>. lOdO

The addition of copper salts to a diet containing a carcinogenic nzo dye nfrords
digree of protection against hepatocarcinogenesis to rats consuming "'J' ' ’

iQEfii Tt has also been found that rats which arc fed .l-meth>l-i-

iHowell 19581. it nas aiso ueen luunu vimu — .

dimethjWinoazohenzene (Neish, 19596) or which receive _smgle mtrajientonca
injections of various hepatocarcinogens (Neish, lOaS, ino9a) suffer a marked
decline in the serum levels of total copper and of the coppcr-contammg enzyme
para-phenylenediamine oxidase (caeruloplasmin). The degree and jicrsisfcnce
of this decline appear to be correlated closely noth the carcinogenic potency of
the injected materials. These results suggest that a dcrangcincnt. of cojiper
metabolism in rat livers may occur at an early stage in hepatocarcinogenesis.

Serum levels of total copper and caeruloplasmin are generally snbnonnnl in
patients with hepatolenticular degeneration (Scheinberg and Sternlicb, 1959)
and Uzman (1957) and Uzman et at (1956) have detected an abnormal copper-avid
ninhydrin-reacting substance, possibly a peptide, in the livers of such patients.
It seemed important to determine whether hepatocarcinogens could induce
the formation of similar abnormal peptides in rat liver. Although work on t his
aspect is not yet complete it has been found that injection of hepatocarcinogens
into rats increases the levels of certain ninlrydrin-positive materials in the liver.

Firstly, it was established qualitatively by paper chromatograpliic and
electrophoretic investigations of phosphate or ethanol extracts of rat liver that
intraperitoneal injections of hepatocarcinogens caused a marked increase in the
level of “ free ” phosphoethanolamine (PE) in this tissue. In general, a strong
hepatocarcinogen evoked and maintained a higher level of PE for a longer time
than did a weaker one.

Secondly, it was found that the livers of rats injected with hepatocarcinogens
contained one or two unidentified ninhydrin-positive substances X and Y which
migrated towards the anode during paper electrophoresis at pH 8-6. Snot X was
0 iscrved regularly after the application of a strong hepatocarcinogen and its
intensity seemed to run parallel with the intensity of PE. Y was not alwsvs

seems to be closely related to glutathione. ^ ^ ^ ^ ^

In the present paper we shall discuss in detail the effect of V.AT,of
on the content of free PE in rat livers In a fot„r! “ .^®P®<^ocarcmogen8
and nature of the peptide-like materiais X and Y wiirbe'?^'””) ?°«"’’rence

to similar substances which have been fminrl ^ tb v ^ ‘^'’^isi^ered m relation
and in tumour extracts. “ tumour-bearing rats

738

W. J. P. NEISH AKB AMT EYLETT

EXPERUIENTAL

Adult male albino rats received single intraperitoneal injections of the azo
djres 3'-metliyl-, 4'-meth3^]-, 2-nietli3d- and 4'-ethyl-4-diraeth3daminoazobenzene
at dose levels corresponding on a mole for mole basis vdth a standard injection
of 16-5 mg. of 3'-metIiyI-4-dimeth3daminoazobenzene (3'-MeDAB) in 0-6 ml.
of arachis oil per 100 g. of body weight, as used in earlier work {Neish, 1958^
1959o) on copper metabolism. At this dose level, 2-meth3d-4-dimeth34ainino’
azobenzeiie (2-MeDAB) proved to be rather toxic and about half of the ara'mais
died within 2 da 3 '’s of the injection. Other rats received intraperitoneal injections
of the hepatocarcinogens tannic acid (5 mg./lOO g. body weight (b.w.)), dime%h
nitrosamine (2-5 mg./lOO g. b.w.), DL-etluonine (11-3 mg./lOO g. b.w.), and of the
supposedly non-carcinogenic carbon tetracliloride (50 mg./lOO g. b.w.) suspended
or dissolved in aracliis oil (0-6 ml./lOO g. b.w.). Control rats received injections
of aracliis oil onl 3 ’- (0-6 ml./lOO g. b.w.).

At intemmls after injections, control and experimental animals were killed
with ether, bled from the Jieart and the livers perfused via the portal vein with
chilled normal saline. The livers were removed, weighed and stored at once at
~ — 15° C. Not later than 2 hours after perfusion, 2 g. portions of the frozen
livers were homogenized uith 4 ml. of full strength Sorensen phosphate buifer
at pH 7’2 in an all-glass homogenizer cooled in ice-water. Supernatants were
obtained b 3 ^ centrifugation of the homogenates for 2 hours at approximatel 3 ^
4500 r.p.m. and 40° F. in an International Portable Refrigerating Centrifuge
Model PR^l. The supernatants were stored at — 15°C. and 0-01 ml. aliquots
were used as required for paper cliromatograpliic and electrophoretic studies.
These extracts seemed to be stable indefinitely if stored at —15° C.

Alcohohc extracts of livers were prepared b 3 '^ homogenization of 1 g. portions
of the frozen livers with sufficient ice-cold ethanol to give a final supernatant
containing approximatel 3 '^ SO per cent alcohol. In order to assess the amount
of absolute ethanol to be added, liver dr 3 '' weights were determined b 3 ' dr 3 'ing
1 g. portions of the livers over sulphuric acid in vacuo at -f 4° C. to constant
weight. The alcoholic homogenates were centrifuged for 10 minutes at 2000
r.p.m. and 0-01 ml. aliquots of the supernatants were used for paper chromato-
graphic studies.

Ttco dimensional paper chromatography . — Separations were carried out on 20
cm. squares of Whatman No. 1 paper in the solvent S 3 'stems (ascending) recom-
mended by Bowden (1959) : in the first direction, 80 per cent phenol with an
ammonia atmosphere (sodium C 3 'anide was not included in the tank) and in the
second direction, a mixtirre of w-butanol (100 ml.), metltyl ethyl ketone (100 ml.),
dic 3 mIohex 3 damine (20 ml.) and water (47 ml.). An aliquot (0-01 ml.) of liver
extract was placed on the paper at a point 3 cm. from adjacent edges.

In early experiments, the papers were stitched with thread in the form oi
cylinders ndiich were allowed to stand in ~ 80 ml. of developing solvent in htre
beakers m an enclosed glass tank. Later, a duralumin Datta frame (Bowden,
1959) was used for multiple separations but this proved unsatisfactory in the
phenol run owing to serious contamination of the papers vdth coloured products
which apparently arose through the action of phenol on the metal frame. A
modified Datta frame which functioned satisfactorily was constructed as iol ows .
two end pieces, H-shaped, were made of glass rods of suitable length and thickness

raOSPHOCTHAXOLAMlKK IN l.lVKUS Ol' P.A'VS

connected wth a pair of brass X X blorUs t<. fonn n fnumv

placed and 4 nan-ow glass rods ^vcrc^PP0llun^^^^ ^

A dozen cbromatograpliy papcn, (-( - - •) ^ M-parati'fl fr-ni

for use sriH, the Datta frame svcre plaecrl on he „ ,.„.

one another by pairs of poreelam peme.mn e ., s Af ’ h ),

treated rvith a solution of ninhydnn (n-2 per ..,,,,1

of rvhieh was added 4 ml. of gincal acol.c acnl. * " , X,ri In ive sonls

heated for 10 minutes at 0. Ponnancnl roeonls of n.nht.lnn |

wrp made ndtli auto-nositivc document paper Xo. 44.

Phosphate buffer smalts did not markedly disturb tlm soparalmn of I""'!'
acid mixtures in this chromatographic system but pmhMU or P”!-'!’;;
stituents of liver phosphate extracts seemed to inter er( ui i ” ‘ ‘ 1 ‘ ‘
substances nith high Ef values in plicnol. More sharply de nied elivomato,i.r.un
Avere obtained rvhen alcoholic extracts of liver were used and t bis nietbo.l pro\ e<t
most useful for comparative studies of the PE content of livers.

Pap(.r ehdrophoresis . — Usually 4 X <*4)1 ml. nliqiiol.s ofjiiei ]>liosj)!i.il< <x-
tracts were applied at the luid-liue of a .strij) of Wlintiimii Xo. 1 paper (1_ - .»'*
cm.) previously moistened vnth pH 8-6 barbitoiic buffer (<><•« ml. of a solnlion
of sodium barbitone (10-3 g./Utre) plus 400 ml. of n .solution orjiarbitone (1-s.-.
g./litre) and blotted. The paper was supported as an inverted \ by a glass rod
under the mid-line in a modification of Durnmi’s apparatus (i)urruni. lO.'di).

The ends of the paper dipped into glass tanks containing ~ 00<) ml. of ]dl sul
buffer. Electrical comiection was established by KCl-agar bridge.s (3 jier cent
agar agar in saturated KCl) connecting the main buffer com])artiiients to small
glass compartments filled with buffer and provided with carbon elect rode.s.
Current was supplied by 3 x 120 volt high tension batteries in series. .Xfter
3 hours, the paper was removed, dried, and ninhydrin-rcacting sjmls were rovealeil
by sprajing with the ninhydrin-acetic acid mixture mentioncfl above. 'I'be
papers were then dried in an oven at 120’ C. until the spot due to aspartic acid
which had initiallj' a distinctive blue colour began to turn reddisi) (~ "> minutes).
By this time spots X and Y if present appeared. In earlier work, tlie ])nj)ei-s
were sprayed with a solution of ninhydrin (0-2 per cent w/v) in 7 i-butnnol con-
taining no acetic acid. Under these circumstances X and Y usually became
c early discernible only on the day following spraying and heating.

'urther analysis of ninhydrin-positive spots was carried out on scction.s
aLh electropherograms run simultaneously. Ninhydrin-positive materials
freoL a 1 IT AAith deionized water. The aqueous extracts were

.ntut taken into a small volume of deionized Avater and the

>t.ons subjected to two dimensional chromatography a.s described Hiovo

little o7r!„ter£l^

.aminn o!.-' components present in the extracts. Likewise mixtures nf

' I s dissolved m barbitone buffer separated Avell in Bow'deii’s system

KESULTS

>'«. 1 »l,o,v. two dimeLlel »fI>“‘'>«cinogem,.

-irtett of livets fw,„ eett injected with ^A) at.„L? oLr

740

W. J. P. NEISH AND ANN RYLETT

(C) 4'-EtDAB. The livers were obtained 6 days after injection. According to
Miller, IMiller and Finger (1957) 3'-MeDAB and 4'-EtDAB are equally potent
carcinogens. Each substance caused about the same degree of accumulation of
PE in rat liver. When synthetic PE was added to the normal liver extract, the
chromatogram showed an increase in the intensity of the rather weak spot due to
naturally occurring PE.

Two dimensional eliromatograms (frame method) of phosphate extracts of
livers from rats (A) 3 days and {J3) 6 daj's after carbon tetrachloride injection are
shoAA'n in Fig. 2. A reference chromatogram (C) is included to show the separation
of PE from a synthetic mixture of several of the amino acids regularly encoun-
tered in the liver extracts. Note the higher level of PE in the 3 -day CCI 4 liver

EXPLANATION OF PLATES
List of nbbreviatioHS for ninin-drin-positive spots :

PE = phosphoethanoinmine.

GLU = glutamic acid.

GLY = glycine.

ASP = aspartic acid.

T.4U = taurine.

G = glutathione.

Vertical and horizontal arrotvs intersecting at the origin of 2-dimensional chromato-
grams indicate the direction of flow of aqueous phenol and of butanol-methylethylketone-
dic 3 'olohexyinmine-wnter solvents respectiveh’.

Fig. 1. — Two-dimensional chromatograms of phosphate e.xtracts of livers from rats 6 daj's
after intraperitoneal injection of

A — arachis oil onlj-.

B _ 3'-MeDAB in arachis oil.

C — 4'-Et0AB in arachis oil.

Fin. 3.- — Two-dimensional chromatograms of phosplmte extracts of rat liver.

A — 3 daj’s after carbon tetrachloride injection.

B — 6 daj'S after carbon tetrachloride injection.

C is a reference chromatogram showing separation of a mixture of the indicated amino
acids from 0-005 ml. of a solution containing 1 mg. of each substance in 5 ml. of phos-
phate buffer pH 7-2. Each spot equivalent to 1 pg.

Note the double spot due to glutathione.

Fig. 3. — Electropherograms (Whatman No. I paper, pH 8-6 barbitone, 360 volts, 3 liours) of
phosphate extracts of

A — rat liver 3 daj-s after tannic acid injection.

B — rat liver 3 daj'S after arachis oil injection.

C = 2-dimensionnl chromatogram of spot 4 from electropherogram A.

D = 2-dimensional chromatogram of spot 4 from electropherogram B.

Electropherograms (as above, 2 hour run) of phosphate extracts of
E — rat liver 0 days after 3'-MeDAB injection.

F — rat liver 6 days after 2— MeDAB injection. ^

Q — j-at liver 6 daj^s after injection of arachis oil onl}'.

Flo. 4. — Two dimensional chromatograms of ethanoiic extracts of rat livers obtained 3 days
after injection of

A — arachis oil onlj'.

B — 3 -MeDAB.

C — 4'-MeDAB.

B — 2-M6DAB.

British Joi'rn'ai- ok Can'ceu,

Vnl, Niv. N-. I.

iitid Kvlott.

British .Toi'rn'ai. or CANfHii.

V..1. NtV. N"

origin

Bkitlsh JoriiSAi. nr (’.\N( rr..

PHOSPIIOETHANdl.AMlNT. IN I.Ivr.liS or It ST ‘-

as compared with tlic O-day CCl^ livor. TIi.' latt-'r -

practically identical mth tho?c given hy exlraels of n lOilay HI, hv't -•■d
of a normal liver. Although carhnn tetraehhiride b f.iid to Ik- n-es v
for rat liver, it resembles the feeble carcinogen I'-MeDAl? in tlm! h '".!) ful.-tA’.*- • -
have a small capacity for increasing the “free" IMC. eonlrnf of t-^t !or: ■■

after injection. ■

Electrophoretic separations of phosphate extn»rts nf ii u>>Tnv;U f.it Ir. <■: .'/>■
and of the hver of a rat injected :i days pn'viously wilii t-annie orjil (.{^ .-.tr. -A:..*-:,
in Fig. 3. Spots X and Y arc present in the tannic .acid liver evlT.-i-'t *. e,
in the normal liver. Spots marked -1. o and nere shown to r.iu-io. pj:,
glutathione, glutamic and aspartic acids respectively.

Paper strips corresponding to spot -1 for normal and t-annie arid liwr -j.-.r.-,
tions were cut from 3 parallel sepanrtions for each ^•\tr.u't and thi' rln:«-.i
hydrin-positive materials were suhjected to t wo-dinu'nKi<,Mal clironnt-gf
Fig. 3 (C) shows the marked accumulation of I'K in the liver of the n; jV,rr'->Ai
mthtanmc acid as compared with the normal nit liver {!>) I'r* 't {f r -■ d
h) are electropherograms showing the oeeurnmee of X in r.U liV.-r ri-,-' •
alter injection mth the powerful carcinogen :t'-Mel)An. Sis.i X j,.
mthehver of a normal rat (G) or in the liver of a rat r, .lav,-^ after in,e,-ti.e c
^ hT2«pd »'»''.vtlrin-’podiive v,p„t:, X o-;d N

™ be discussed m a separate communication.

PE duepiepatltctyft^^^^^^^^^ hny’’'!''’l K., l'”'"

carcino°enicl xitJ i! ^ '»ul (/;) g.Mel)\|? i'. ..

»f PE due to the feebly earcinogotiic d'-Mel) li, . T"'

**.1 1„„ ,e„l3 pj, g™« d .'loDAl 1 tlnv., „r„.r injeeti,,,,.

ths wtasnce (Table I). ‘ 'Iriya iirior .ij

P^tluced a decrease in th? f ‘'™°genic but not of non c-ne; df-l)

J'^'-'^ted levels of PE aJd ^ In^ nerd 'l.v.-s

weight percental t /PPearance of X narallol H • ’ T"' ‘In*

?«P‘''tocarcinogens. to note that n.fl in liver

weight percentaae Tr /PP^arance of X parallel tr ’ T' ‘Ik*

"‘''’’'>'»ogcnie sub'

742

AV. J. P. NEISH AND ANN RYLETT

Table I. Effect of Various Hepatocarcinogens and Related Substances on the
Dry Weight Percentage of Rat Liver

Substance

Time after

Liver

dry weight percentage
in experiment

injected in
arachis oil

injection

— A-

(days)

' 1

III

Nil

20- 0'

26-1

29-2

30-0

29-1 (4-8)

20-8

25-4

27-5

28-6

27-2 (4-6)

2G-3

—

26-3

31-0

2fi-7

—

3'-MeDAB

22-2

23-8

22-4 (2-9)

22-7

24-1

—

21-8 (2-6)

26-6

26-0

—

4'-MeDAB

20-0

26-1

27-6

27-4

—

2-MeDAB

24-0

2S-0 (5-1)

—

. —

—

25-9 (5-7)

27-4

—

4'-EtDAB

■

23-7

—

20-0

—

Tannic acid

■

26‘0

___

22-n

—

26-7

—

DL-ethionine .

29-9

27*2

—

24*3

—

Dimethylnitrosamine

23-0

25-7

—

28-1

—

27-4

—

Carbon tetracliloride

28-6

—

32-4

—

29-1

—

* Figvires in parentheses, liver as percentage of total body weight.

It is interesting that marked accumulation of PE occurred in the case of
DL-ethionine only at the 12th day after injection at trliich time the decrease in
liver dry weight percentage was most marked.

Although elevations of the level of PE is the most prominent feature m
chromatograms of extracts of carcinogen-treated rat livers, changes in the levels
of other ninhydrin-positive substances do occur. For example, 3 days after
injection of the azo dyes (Fig. 4) the following changes were obsen'’ed :

Substance

injected in
arachis oil

LeveU of
glutamic acid

Glycine

Taurine

Nil

—

-f —

3-MeDAB .

■*” "T

4'-MeDAB

—

-4-

2-jMeDAB

—

_i 1_

-r

PHOSPHOETHANOI.AMIN'K IN I.lVKltS OK K.ATS

Table II. — Rclalwc PE Coniculs oj Pnl Liver tit \’iirit)U.^ Tiineji After liijerjinn
of Corrinogciiic nr Xnn-cnrciiurjcnic Sutn^laneex

Substance
injected in
arachis oil
3'-MeDAB .
4'-EtDAB .
4'-3IeDAB .
2-JIeDAB .

Tannic acid .

Dimethj'lnitro.saniine

DL^thionine
Carbon tetrachloride
Nil

Knirini).

penieity

I^vrl Ilf J'liii’liliii^ttinnidntmnr'
nltiT itijivtiiin*

(nt (ln\ o|

ment '^^“^S^uL’itv '"coring «-n^ "'"n ""i'-. Knr tl.-

injection time. ' ’ ‘ viminlly. At lenM Ovo livrra m/to rxotnitinl fur onrl,

detSk ..f nlmdudic rt,rnr,i„„ i. in 1, nr.lv

P« hepatocarobogm in anito of .1 r r nf

^° ■’ areobser^•edincltracts of the,o^ivo« {"M" ‘ KrT..n,n...,t VK

the normal level tvh c^ ’"'i""'’'.’''-

of 1' mg./ioo g. svet weight of liver.' *” Awn, mm. Imndun nnd K‘„,.r«t (Hb'iD) ia of the onh-'r

»ci.l n„.l ^Ivcinc

of the livers was annrpoioKi t^ tjiese snb.stfincc.s, but (ho (aiin’no ottntoiil
I.»b%fcp,eLS llml cvslri,,,. „S1,

discussion

Sath j."*' ''7” "-W- liepaloci,roi,.ogo„a

i--:5;KXS

possibility i” njamniaiian tissues tvftl. ^ pllosphatidyl-

PE “il Wota (1956) shoS that elfv''®.”'* «“ “Pcoiid

to c •if'' 3'-MeDAB^“^ ? accumulation of Sine

744

W. J. P. N'EISH AND ANN RYLETT

Another possible explanation for PE accumulation may be found in the
suppression of liver alkaline phosphatase activity by hepatocarcinogens. Accord-
ing to McCanee, Morrison and Dent (1955) and to Eraser, Yendt and Christie
(1955) patients with hypophosphatasia excrete PE in their urine. This is attri-
buted not to renal malfunction but to a lowered serum ahraline phosphatase
activity resulting in accumulation and excretion of PE. According to McCance
et al. (1955), PE can act as a substrate for alkaline phosphatase. Sachs, Baumer
and Menldiaus (1959) found by histochemical methods that oral administration
of the hepatocarcinogen tliioacetamide to rats does indeed lead to a decrease in
liver alkaline phosphatase and Tsuboi and Stowell (1951) observed a decline
in the alkalme phosphatase activity of mouse liver following a single feeding of
carbon tetrachloride (carcinogenic for mouse liver). Woodard (1943), however,
found a marked elevation of alkaline phospliatase in the precancerous livers of
rats fed d-dimetlijdaminoazobenzene.

Since serine is a probable precursor of ethanolamine and PE it was unfortunate
that in the present study certain limitations of the clu’omatographic technique
prevented us from followmg easily the fate of serine and ethanolamine in rat
liver subjected to the action of hepatocarcinogens. It is hoped to complete tliis
aspect of the work. Meanwhile it may be noted that Levjq Montanez and Dunn
(1955) reported a decrease in the serine level in rat liver after a single massive
intraperitoneal injection of the hepatocar-cinogen, DL-etlrionine.

Kensler, Bierman and Condouris (1955) reported that the addition of ethanal-
amine to a diet containing 4-dimethylaminoazobenzene afforded marked protec-
tion to rats against hepatocarcinogenesis. In view of our findings it would seem
that ethanolamine supplements can overcome some deleterious action of a hepato-
carcinogen wliich is expressed in the accumulation of PE in the liver.

In certain other aspects of liver cancer, peculiarities in ethanolamine meta-
bolism have been noted. Thus Reid, Landefetd and Simpson (1952) have shou’ii
that the ethanolamine moiety of the choline of rat liver tumours is apparently
s 3 aithesized in a different waj'' from that of normal rat liver. Dent, Fowler
and Walshe (1951) ha^m observed the excretion of large amounts of ethanolamine
by a patient with primary hepatoma. Tliis metabolic defect was considered to
be either cause or consequence of the hepatoma.

It is of interest to recall the statement by Haven and Bloor (1956) that ab-
normalities in the metabolism of ethanolamine if established as directly concerned
in carcinogenesis by azo dyes would implicate phospholipids in the induction o
hepatoma.” Because of the close correlation which has been observed between
the hepatocarcinogenic potencj'^ of a substance and its ability to cause accumiila-
tion of PE in rat liver, a more detailed biochemical study certaiidj’^eems warran e
of the metabolism of phospholipids, serine, ethanolamine and PE m the stages
of hepatocarcinogensis.

siraiJiABY

Single intraperitoneal injections of powerful liters

albino rats caused a marked accumulation of p substance Iiavino- the

and the appearance of an unknown ninhydnn-pos.tn e substance i.a.ing the

properties of an acidic thiol peptide.

PHOSl'HOKTHANOhAMIN’K IX I.IVKUS OP HATS

ADDKN'nilM

Since rating this our attention lias been direct ed to several relerenees wliieb
may have a bearing on our investigations.

(a) Ferrari and Tenconi (IdaT) stated that the livers of normal and adrenalee-
tomized rats ivhich had been injected repeatedly u’ith 10 per cent ethanol contained
respectively S-O and 23-6 nig. of free PE jier 100 g, of fresh tissue.

(b) An increase in the free PE content of (he livers of rats subjected to ailrenal-
ectomy had also been noted by Awapara, Skcllcnger and Man/. (lOaf)).

(c) Spicer and Weise (15156) observed an elevation of PE in the livers of rabbits
and gmnea-pigs which had been exposed to X-irradiation.

to (a) and (b) it may be noted that DaVair/.o and Eversolc (105S)
Burgojnic (15154) found that total adrenalectomy
PT 7 fJ ■ azo dye carcinogenesis. Perhajis the transient, rise in livi«r

norarv mjection of an hepatocarcinogen is to be explained by some tem-
P } deletenous action of the carcinogen on the adrenals.

REFERENCES

W •/. b!ol. Chan., 183. .545.

Boi^kTh nqsQ'i if '-Tex. Jicp. Biol. Med., 13. 1.

Dekt, C E Forann ^ 8 ' "«5-

DimBuii, ElSSoI j 1 (1»51) Biochan. ./., 48. xiii.

Feebidt V C^em. Soc., 72, 2943

h*™k, r, L LTblook w °e TS L"- /.»«»/, i. 2 sr,.

Homttll, J. S— (19581 rAv Res., 4. 238.

KEMfEDY p p J. Cancer, 12, 594.

{^5'’slek,’c. 222, 193.

Mon™ ’g ■ fn ^—(1955) J. nal. Cancer Jnsl., 15. 15G9

IcCance, R. a., £Son 212. 985.’

JlHiER, J. A.,yitLLEK E o’ E— (1955) Lancet, i, 131.

^Ksh, W. j p — (19581 'fttipy- — (l^o7) Cancer Bes. 17 387

Ua. ?■ . «. 287.-|1959a) lUi., IS, 20.-(i!S, IIU., 15,

JW”! J.'cttaMrairK’ *2. ^19-
f C. A.-(195I)kJ/Et.?r6?. '•

> riCER, s. g Weise V ^^^59) Gastroenterology, 37, 550

3'-m^.onidis, A., A™’ 77^1956) Fnzt/molopta, 17, 263. ’

805. ’ Burgoyne, P. H.— (1954) J r,

Tsunoi K K c. > a. u»04; j. nat. Cancer Inst., 14

^ UJ43) Cancer Res, 3 , 159 ^

746

THE EFFECTS OF SOME CHEMICAL CAECINOGENS UPON THE
-SH LE'^CELS OF TAKGET AND NON-TAEGET TISSUES

G. CALCUTT, D. DOXEY and JOAN COATES

From the Department of Cancer Research, Mount Vernon Hospital, and
the Radium Institute, Northiuood, Middlesex

Received for publication October 29, 1960

The role of sulphydryl (-SH) groups in both tumour growth and the induction
of tumours has been a subject of interest for many years. Actual experimental
work in this field has been limited and often only via indirect approaches. Methods
of measuring the -SH within tissues have been unsatisfactory and in many cases
involved tedious procedures with specialised equipment. Recently, Calcutt and
Doxey (1959) described a simple technique for tissue -SH measurements using
small Aveights of tissue. This technique was then used to determine the effects
of various carcinogenic and non-carcinogenic hydrocarbons on the liver -SH
values of mice previously treated Avith one of these hydrocarbons (Calcutt, Doxey
and Coates, 1959). This Avork dealt with the relationships of -SH levels to the
metabolism of the agents in question, since all are knoAvn to be metabolised in
the liA'^er but none can be regarded as a true hepato-carcinogen.

The present paper records a study of the effects of knoAvn chemical carcinogens
on the -SH levels of susceptible and non-susceptible tissues. We have been
influenced in this choice of subject by knowledge of two previous papers which
bear upon this field. Bo5dand and MaAA'Son (1938) found that intraperitoneal
injection of 3 : 4 : 5 : 6 dibenzocarbazole (a knoAvn hepato-carcinogen) causes a
considerable rise in liAmr glutathione AAdiich is persistent over several months.
The IWers of mice injected with methylcholanthrene or 1 ; 2 ; 5 : 6 dibenzanthra-
cene (neither being a liAmr carcinogen) did not show a similar increase m
glutathione. Di Paolo and Niedbala (1957) found that after a single painting Avim
either 1 : 2 : 5 : 6 dibenzanthracene or 9:10 dimethylbenzanthracene the -oH
content of mouse skin was raised aboA’^e the normal Amlue and this increase aa'ss
persistent for at least fiA'^e days. The chemically related, but non-carcinogenic
compound, anthracene, AAms found to haA’^e no effect on skin -SH leAmls.

The Measurement of Tissue -SH

As the technique used has been described in detail elsewhere, only an outline
Avill be given here. KnoAAm Aveights of slices of the tissue under examma ion ar
immersed in a ImoAvn volume of a standardised 5 X 10“® M solution ^
mercuribenzoic acid (C.M.B.). After a time interval (approximately 3 ,

for reaction Avith the -SH groups to occur, the unchanged C.M.B. is i r
potentiometrically Avith 2 x 10 “‘‘m cysteine hydrochloride. The im lyo ™ .
originally used in developing this technique haA'^e now been replaced y
ised titrimeters based on the design of Stock (1958). These
superior to millivoltmeters in use as settling times are negligible an en j

CHEMICAL CARCINOGENS AND — SH LEVELS

747

deflections are large and easily read. During the present work every
has keen mn in duplicate, the two runs being done nt the same time on .separate
sets of instruments End points on the two sets of ccpnpmcnt have
completely in agreement or within 0-1 ml. of one another. I he mean of (he tu
resufts has been used for calculation purposes. The tcchnupie has been ndajjtc
for glutatliione estimations. Weighed amounts of tissue have been ground m
a few ml. of 10 per cent metaphosphoric acid and centrifuged. The snjicnmtant
together with a further washing of the tissue has then been neutralised to pH
7-0 with 5 per cent caustic soda solution using IJroino-cresol jiurjile ns an indi-
cator. This solution has been treated for -SH measurement as in the case ol
tissues. The neutralisation of the acid solution of glutathione is essential ns
C.M.B. is insoluble in acids.

The weights of tissue used for estimations have normally been in the range
80-200 mg.

Expcrimcnlnl

3 : 4 Benzopyrene . — One hundred and seventy strong A male mice were divided
into two groups. The animals of one group each received a once weekly applica-
tion of 0-2 ml. of a 0-5 per cent acetone solution of 3 : 4 benzopyrene. The other
group were used as controls. Daily for the first week and then at intervals of
seven days five animals of each group were killed. Small pieces of skin (after the
fur had been clipped) were removed from the painted areas or from the correspond-
ing areas of the untreated mice. Estimations of -SH were then made on the
pooled samples of skin. The results over a period of 6.5 days are shown in Fig. 1.
Since, over this period, no pattern has been detectable in the levels for the control
skins these figures have been shown as a mean figure and a standard deviation.
All measurements are expressed in terms of /<g. of -SH per 100 mg. of rvet Aveight.
of skin.

After the first treatment AAuth benzopjwene the -SH level of the skin rose
and then declined over the first wnek to a normal level. Further treatment gave
enhanced levels for about 6-7 weeks when a return to the normal level occurred.

It has been shown by Calcutt and Powell (1947) that after skin painting of
polycyclic hydrocarbons a large amount of the applied reagent is removed by the
animals licking themselves. The agent removed in this fashion is carried down
re gut and is transferred to the internal organs of the body. AVe have, therefore,
one -SH measurements on both the liver and the kidneys of both control and

choice of these two tissues is based on the evidence
0 eigert and Mottram (1946) that benzopyrene is metabolised in both, but that
of^S*- 4 evidence for either being susceptible to the carcinogenic activity

of results obtained with liver are shown. No definite indications

Pic I w changes were obtained. The kidney results are shoAvn in

there was no evidence of specific changes in -SH levels.

W.-cfo (2 acetylaminofluorene ; 2.A.A.F.). — ^Fiftv female

"P ^vith wafer to ^ ^ ^^s

-to ofV;trcl^ to added ft the

found to br^Tentahle'f f f ^^s fashion

acceptable to the animals and the control group ate and ere^

was
grew as

748

G. CALCUTT, D. BOXEY AND JOAN COATES

:10

Number of Ireatmenfs with carcinogen
>11 1 1 1 1 2 3 4 5 6 7 «

• •

• • •

1 2 3 4 5 6 7

16 23 30 37 44 51 58 65

Days after commencement of treatment

Fig. 1. — The effects of treatment with 3 ; 4 benzopjTeno on mouse skin -SH levels.

In this and all further figures, the mean eontrol value is shown ns a henr’j' line and the
standard deviation by the dotted area. Experimental figures are shown ns filled circles.

Fig. 2. — ^Mouso liver -SH values from animals which had been painted with 3 : 4 benzopyrene.

CHEMICAL CAKCINOOEXS AXII --.SH LEVELS

745)

normally. The experimental group were found to cat. slightly less than the

controls. • cu

At weekly intervals one animal from each grouj) was killed and liver -SH
levels determined. The results are showm in Fig. 4. As no iiattern was found

I— I tilt ■ ........ t

1 2 3 4 5 6 7 9 16 23 30 37 44 51 58 65

Days after commencement of treatment

Fig. 3. Mouse kidney -SH levels from nnimnts wliicli lind been pninted svifli 3 : 4 benzopyrene.

‘^■'^Perimental resu ts a^e standard deviation have been calculated and the

tained two excentiona^t t ^ ^he control figures con-

standard deviation^ miSh 'b ^ and

'calculation of a mean and sta^f^^d otherwise he the case. Re-

n and standard deviation, omitting these two very high

750

G. CAiCUTT, D. DOXEY AND JOAN COATES

values, gave lower figures which were in close agreement with another set of
figures independently determined upon a similar group of rats for another purpose.
The amended mean and standard deviation have also been shown on Pig. 4 .
Whichever figure is taken as representative of the mean control value the results
indicate an elevation of liver -SH over the first 7-8 weeks of feeding the carcino-
gen. Values then return to near the control value.

Hartwell (1951) and Shubik and Hartwell (1957) in their survey's of agents
tested for carcinogenicity list several references to the induction of kidney tumours
by 2.A.A.F. We have, therefore, also examined the kidney -SH levels from the
same rats as were used in the previous experiment. The results are shown in
Fig. 5. The picture, although not well defined, is suggestive of a rise in kidney

* * * * _* ' ■

6 13 20 27 34 41 48 55 62 69 76 83 90 97 104

Days after commencement of treatment
Fig. 5. — The effects of feeding S.A.A.F. on rnt kidney -SH levels.

-SH during the first six weeks of the experiment and then a fall to below the mean
control value.

As a tissue which is not susceptible to tumour induction by 2.A.A.F. we have
taken cardiac muscle (from the ventricles) of both control and treated animals
The results are given in Fig. 6. The picture here rather resembles that obtained
with the kidney.

p-Dimethylaminoazobenzene {dimethyl yellow ). — This agent is a potent hepato-
carcinogen in rats on a poor diet. Fifty female Wistar rats aged twelve wee 's
were divided into two groups and given a diet of rice and water supplemen
with carrots twice weekly. After ten days on this diet one group had dune y
yellow in olive oil added to their rice at the rate of 0-6 per cent of the ^et. le
control group had a similar amount of olive oil added to their rice. At wee ' J
intervals commencing six days after treatment began, the liver -SH leve va
measured on a rat from each group. The results are shown in Fig. 7- s
control figures varied wudely^ — ^possibly as a result of dietary effects no me

CHEMICAL CAUCINOOENS AND

-Sll LEVELS

7r)l

fitrure has been calculated, but. each pair of rosulls has been jilo tied. Between
27 and 48 days after commcuccnicut of trcatincnt. the expeniuenlai liguros woie
above the controls, thereafter the iiosit.ion was reversed.

13 20 27 34 41 48 55 62 69 76 83 90 97

Days after commencement of treatment

Flo. 6. — The effects of feeding 2.A.A.F. on rnt enrdiiie imisele -SH levels.

104

The effects on liver -SH values of feeding p-dimethylaminoazobenzene to rat.s on a
poor diet. Control values are shown as crosses.

"STT

results have also been made on the kidneys of the same rats. The

althouoL individual readings have been plotted,

apuei^ tn 1^ nuctuations in the control levels are only minor in nature. There
to be httle distinction between the two sets of figures.

752

a. CALCUTT, D. DOXEY AND JOAN COATES

M/ii/l carbamate (tiretliane).—S‘oTiy female Strong A mice aged 10 weeks
were divided into two groups of 20 each. Both received the normal pellet diet as
food whilst one was given tap water to drink the other received a 0-1 per cent
solution of urethane in tap water. At weekly intervals commencing 6 days after

20l*

c/2

“ loH

+ + + +

+ •

• ^

+ +

6 1.1 20 27 34 41 48 55 62 69 76

Days after commencement of treatment

Fig. 8, — The efieots of feeding dimethylnminonzobenzene on rat kidney -SH levels. Control

values are shown ns crosses.

I ■ • ■ ■ * « ._ ■ I I i —

6 13 20 27 34 41 48 55 62 69

Days after commencement of treatment

Fig. 9. — The effects of drinking 0- 1 per cent urethane on mouse lung -SH levels.

the transfer to urethane one mouse of each group was taken and the - ' ^

for the lung tissue were measured. The results are shown in Fig. 9,
it appears that the experimental animals show a tendency to an eleva e

level, as compared with the controls. , . i. i, a rocpived

It was found by Haran and Berenblum (1956) that mice which a
urethane systemically did not normally produce skin tumours, but a

CHKMICAL CAKCINOOKXS AND Sll DKVKI.S

would occur following further treatinont with croton oil. We hove, theiofoie.
estimated the -SH levels in the skins of mice nse<l in the previous ex])ernne,nt .
The results are given in Fig. Kh from whicli it is seen that then* is a ]ier.‘’istcnt.
decline in -SH values as conij)ared with the control values.

Fig. 10. Xhe effects of drinking 0- 1 per cent urctlmno on n\ou?o skin -SH levels.

*— * « . ■ . » 1-1 I t - ■

4 11 18 2 5 32 39 46 53 6 0 67 74 81

Days after commencement of treatment
Fig. 11. — effects of stilboestrol on hamster kidney -SH levels.

— ^Twenty male hamsters aged eight weeks each received a sub-
was pellet of stilboestrol. A similar untreated group

arm • Starting four days after the implantations one of each

ore s? '' weekly and the kidney — SH levels were measured. The results

it slialTl^ 1 ^^ ^ ^ ' ^^i^^rally, the experimental animals have tended to show

notw ~SH value than the controls. This experiment, however, has

It \VT satisfactory as the absorption of the pellets has been very variable.

' ^ *^°i'ced that in the case of the two high readings (39 and 74 days) the

754

G. CALCUTT, D. DOXEY AND JOAN COATES

implants had almost disappeared, but in some of the other animals their appear-
ance was as when put in. ^

Measurernents of -SH have also been made on the liver tissue ; the results
being given in Fig. 12. Here again the experimental figures fall rather below the
control level.

4oL

4 11 18 25 32 39 46 53 60 67 74 81

Days aftpf commencement of treatment
Fig. 12. — The effects of stilboestrol on hamster liver -SH levels.

The Source of Elevated -SH Levels-

Reference was made earlier to the finding by Boyland and Mawson (1938)
of elevated liver glutathione levels after treatment with 3 : 4 : 5 ; 6 dibenzo-
carbazole. It is obviously of interest to know whether elevated levels found in
some instances in the present work are the consequence of a change in glutathione
levels or also represent changes in protein -SH levels. Glutathione estimations
have been done in a few cases during the experiments with N.2 fluoroenylacet-
amide and p-dimethylaminoazobenzene.

Table I records figures obtained diming experiments with 2.A.A.F. The

Table I. — The Distribution of -SH in /ig.jlOO mg. of Liver of Control Rats

and Rats Fed 2.A.A.F.

Control

2.A.A.F.

Total

Glutathione

Protein

Total ■

Glutathione

Protein

-SH

26-4

12-0

13-9

29-9

13-2

16-7

24-1

7-G

16-5

30-0

12-1

18-5

27-5

10-5

17-0

34-6

10-3

24-3

25-2

14-4

10-8

27-3

16-9

10-4

26-0

14-2

11-8

29-6

15-5

14-1

25-8

11-8

14-0

30-4

13-6

16-8

Mean

CHEMICAL CAUCINOGENS AND — SH LEVELS

755

figures are very limited in number but. do snggc.st that l)otb gintatliione and
protein -SH have been aflFected.

Table II records similar figures obtained with rats fed 7 )-dimethylnmino-
azobenzene together with a rice diet. Here, because of the limited figures avail-
able and the wide fluctuations in levels conclusions arc difficult, but certainly
an impression is gained that both the glutathione and protein Icv'cls are .subject
to increase.

T.able II. — The Distribuiion of -SH hi /ig. /lOl) vig. of Liver from ('ontrol Rats
and Rats Fed ■p-dhncthylamivobenzene

Control

yi-Dimothylaininonzobenzene

Total

Glutathione

Protein

Total

Oliitntliionc

Protein

-SH

20-4

5-3

14-9

27-1

n-0

18-1

15-5

3-1

12-4

10- 0

.7-8

10-8

13-5

il-O

7-r.

ir)-7

7-r.

8-2

DISCUSSION

The work recorded above is part of an extensive programme designed to
investigate the role of tissue -SH groups in the carcinogenic process. In view of
this and the paucity of data in the field as a whole, discussion here is limited to
certain points of immediate interest in relation to the results obtained.

The figures obtained indicate that 3 ; 4 benzopyrene, N.2 fluoroenylacetamide
and urethane are capable of inducing an elevation of -SH levels in tissues upon
which they exert their carcinogenic response. Suggestions of a similar effect
with p-dimethylaminoazobenzene and stilboestrol were also obtained. On the
other hand, the agents used have only shorvn one example of a non-susceptible
bssue -SH level being affected. That is the case of urethane and mouse skin,
here the evidence would imply deletion of sulphydryl as was previously found by
alcutt al. (1959) for the case of anthracene and liver tissue. In this particular
case it IS known that mercapturate formation plays a part in the metabolism of
an iracene, but there is no evidence available of mercapturate formation after
reatment with urethane. However, it may or may not be significant that
leckert (1951) has found an unknown reducing substance in the urine of man
alter urethane treatment.

in ®%ht evidence available indicates that where -SH levels have risen the
both glutathione and protein -SH content. Boyland and Mawson
een 1 increased liver glutathione after treatment with the hepatocarcino-

(194n ■ ^ ® dibenzocarbazole but did not measure protein -SH. Crabtree

varin the glutathione content of mouse skin after treatment with

uierc'^^t polycyclic hydrocarbons. Falls in level were found with the known
dibenJ^ producing agents — anthracene and phenanthrene ; whilst 1 ; 2 : 5 ; 6

^ ^^^zopyrene had no effect. As these experiments were

not Lo 0 ^ 4 . limited time period (up to 4 hours) too much weight should

\^!jfj^^tfcached to these findings.

tile aues'u ™ content have occurred another problem is posed in

Ihione it origin of this new reactive material. In the case of gluta-

cou d arise at the expense of oxidised glutathione by reduction, although

756

G. CALCUTT, r». DOXEY AND JOAN COATES

almost certainly not by the direct chemical action of the carcinogenic chemicals
used. Alternativety it would appear to be new formation of glutathione. In
the case of the cellular protein the increased -SH could be derived from either
one or a combination of the following : breakage of existing S-S bonds to give
-SH ; an actual increase in the cell protein eontent or an apparent increase in
protein content as the result of loss of water. In this connection it may be
pertinent to notice that Barer and Joseph (1960) found that the lymphocytes from
mice which had been treated with X-rays or Chlorambucil (both of which may be
regarded as carcinogenic agents) showed an increased cjdoplasmic protein content
over periods of up to 28 days.

The point still remains as to whether -SH levels are concerned in the induction
of tumours. Certainly'^, the present work is suggestive of chemical carcinogens
being able to bring about a rise in levels in susceptible tissues but not in non-
susceptible tissues. Alongside this must be placed the experience of Crabtree
(1944, 1946) that mercapturate forming agents — bromobenzene, anthracene and
phenanthrene — act as inliibitors to the induction of skin tumours by polycyclic
hj’^dro carbons. Additionally'^, Crabtree (1945) has also found that dibasic acids,
such as citraconic and malonic acids, inhibit -SH groups and also inhibit skin
tumour induction. Crabtree considered the inhibition of tumour induction in
terms of removal of -SH groups necessary for combination with the carcinogen,
but now it may appear more relevant to consider the matter in terms of prevention
of a rise in tissue -SH.

The previous paragraphs have raised many questions which require further
experimental work for their solution. It is hoped to be able to proffer answers
to some of these problems in some future papers.

sinvinrARY

3 : 4 Benzopyrene has been found to induce an increase in the -SH content of
mouse skin, but not in that of mouse liver or kidney'.

N.2 fluoroenydacetamide causes an increase in liver -SH levels in rats. Effects
on rat kidney and heart muscle were ill defined but suggestive of a rise in levels.

p-Dimethydaminoazobenzene caused a slight increase in the liver -SH levels
of rats (as compared with controls) over a limited period. No effect was detected
in kidney's.

Urethane was found to increase mouse lung -SH levels but to depress mouse
skin levels.

Stilboestrol gave some indications of elevating kidney -SH levels in hamsters
but caused a slight fall in liver levels in the same animals. These findings have
been briefly discussed.

The expenses of this work were defrayed from a block grant by the British
Empire Cancer Campaign.

REFERENCES

Basee, R. Axn Joseph, S.— (1960) Hxp. Cell. Res., 19, 51.

Bieckeet, A.-(1951) ges. exp. Med., 117, 10, quoted by Wi hams, R. 1-
“ Detoxication Mechanisms ”. London (Chapman and Hall).

Boyeahd, E. and Mawsox, Elinoe, H. — (1938) Biochem. J., 32, 1460.

CHEMICAL CAnCIXOCKNS AND -~.SH LEVELS

Calcutt, G. axd Doxey, D. — (1055)) Exp. Cell. JIc.'i., 17, r»12.

Wem, Doxey, D. axd Coates, Joan. — (lOS!)) Jiril. J. Cancer, 13, 711.

Idem AXD Powell, A. K. — (1947) Ibid., 1, 323.

Crabtree, H. G.— (lOW) Cancer Ecs.. 4, RSS.— (1045) Ibid., 5, 34(1.— (1 UK)) Ibid., 6, 503.
Di Paolo, J. A. .,and Niedbala. F. — (19.57) Proc. Hoc. exp. Biol. A^)^, 96, 255.

Haeax, Nech.ama AND Berenbulm, I. — (195(1) Bril. J. Cancer, 10, 57.

Hartavell, j. L. — (1951) “Survey of componncl.s which hnve heen tested for carcino-
genic .'ictivity.” 2nd Ed. Bcthc.sdn (Xntionnl Cancer In.stitiitc).

Shcbik, P. AND HARTn'ELL, .1. L. — (19,57) “ Snrvc}’ of compound.s which hnve been
tested for carcinogenic activity.” l.st Supnleinont., Bctlic.sdn (National Cancer
Institute).

Stock, J. T.— (1958) Anahtst, 83, 50.

Weigert, F. and Mottra.m. j. C.— (1940) Cancer Bes.. 6. 109.

758

RELATIVE METABOLIC ACTIVITIES OF HISTONES
IN TUMOURS AND LIVER

J. A. V. BUTLER and D. J. R. LAURENCE

Frmn the Chester Beatty Besearsh Institute, Institute of Cancer Research,
Royal Cancer Hospital, Fulham Road, London, S. fr.3

Received for publication October 5, 1960.

Very little is known about the biosynthesis of histones in either normal or
cancerous tissue. As major constituents of the chromosomes, it might be ex-
peeted. that their synthesis will be closely connected with and possibly controlled
by UNA synthesis. As the rate of UNA sjmthesis is determined by the rate of
cell division, it follows that histone s 3 mthesis maj'^ similarly differ in actively
dividing and non-dividing tissues.

Comparisons between tissues using radioactive amino acids are complicated
by unloionm factors such as the rate of transport of the labelled compound to
the site of synthesis and the dilution by the non-radioactive amino-acid pool
(Loftfield and Harris, 1956). Correct information on these factors may be
difficult to obtain but comparisons of limited significance can be made if the
activity of the C 3 d;oplasmic proteins is used as a standard of reference. Such
comparisons might well show a marked difference between actively dividing
tissues and non-di%iding cells.

An experiment of this kind was carried out by Rotherham et al. (1957) rvho
compared the partition of gIycine-^^0 between nuclear and cytoplasmic proteins
of rat hepatoma and normal rat liver. They found that 20 hours after injection,
the specific activities of nuclear histones were considerably less than those of the
cytoplasmic proteins in both cases, although the ratio of labelling of histone and
cytoplasmic protein (H/G) was greater in the case of tumour (0-5) than for normal
liver (0-15).

Busch, Davis and Anderson (1958) and Busch et al. (1959) made similar
comparisons between the rates of uptake of various amino-acids into the histones
of Walker and Jensen rat tumours and other normal rat tissues, the corresponding
uptake into cj'toplasmic proteins being also obtained. Within 1 hour of injection
the specific activity of the tumour histones w^as comparable with the activity of
the most active cytoplasmic fraction of the tumour (H/C = 1'2). In normal
tissues on the other hand the reverse Avas generally true (H/C = 0-6).

In the original paper of Busch et al. (1958) the liistone was obtained as a
precipitate on neutralising an acid extract of nuclei. It has been shoryn by
Davison (1957), both Avith tumours and rat liver, that this procedme ghms a
complex of histone with an acidic protein. In a subsequent paper (Busch el at.,
1959) precipitation of dialysed nuclear extract with acetone and ammonia Avas
employed which shmdd give a better product. , i

In this laboratory we have developed methods for preparation of lustone avIu ^
are knoAvn to give a product Avith acceptable analytical properties, either >
extraction of the whole histone from isolated deoxjmucleoprotein or by isola i

METABOLIC ACTIVITIES OE HISTONES

75!)

of a well characterised histone fraction b^y extraction in the presence of 70 per
cent alcohol (Johns et ah, 1060). The latter method is so specific that in the case
of calf thjTnns, no initial separation of the miclei is necessary. We have there-
fore repeated some of the -work with our OM'ii methods to ensure that the effects
observed are not due to non-basic impurities.

EXPERIMENTAL

Radioactive /-lysine-U-^''C (specific activity 7-5 mC/mM) was obtained from
the Radiochemical Centre, Amersham. Rats were of CB strain, average iveight
250 g. and mice were CB stock, average weight 30 g. The animals M’ere fed
until 5 hours before the experiments. Injections were intraperitoneal and the
animals were killed 1 hour after injection. Counting ivas at near infinite tluck-
ness on 1 cm^. or 0-25 cm.” planchettes, a small correction being applied for the
actual weight of protein on the planchette. An end windoiv counter was used,
the background being 10-11 c.p.m. measured over 30 or 60 minutes. Samples
were counted for 10 minutes each. Amino acid analj'ses were by the method of
Phillips and Johns (1959).

Histones from rats carrying the Walker tumour {Method A)

Rats, having received an implant of the Walker tumour 7 days previously,
were injected with 5 /ic. lysine each. After killing the animals’ livers and tumours
were rapidly removed and separatelj’’ frozen on solid CO^ blocks, necrotic regions
of the tumour being discarded. Frozen tissues w'ere stored at — 20 ° C. and used
within 3 hours.

The frozen tissues (35 g.) were each placed in enough ice cold 0-9% w/v
NaCl solution (pH 7) to bring the total volume to 200 ml. After standing for a
tew minutes the partially thaw'ed tissues w'ere homogenised for two minutes in
an MSE homogeniser running at maximum speed (14,000 r.p.m.). The nucleo-
protein fractions were obtained by centrifuging at 1500 g for 30 minutes, the
supernatant liquid being retained for isolation of cytoplasmic proteins. Nucleo-
protein was washed tAvice in 0-9 per cent NaCl by repeating the homogenisation
The Avashed nucleoproteins AA^ere each extracted OA'emight
h 60 ml. 0-25 H-H 2 SO 4 at 4° by rocking in vessels containing glass beads.

clarified in a Spinco model L refrigerated ultracentrifuge ( 20,000
T 1 , adjusted to pH 3-8 by addition of solid barium hydroxide,

le a,rium sulphate precipitate was removed by centrifuging (2000 g, 30 minutes).

then obtained from the remaining solution by adjustment to
P and addition of 3 vol. of acetone as described by Davison (1957).

Hsiones from mice carrying the Crocker tumour {Method A)

Avith ' 1 *^ receiAmd an implant of the Crocker tumour 9 days before injection
_r.. 13 'sine each. No necrotic areas AA’^ere found in the tumoms and 25 g.
tissue were treated as described for the AValker tumour.

■^’’ginine-rich histone from rat liver {Method B)

droimi^' tissue from rats injected AAutli 5 /<c lysine each, AA'ere frozen bj^

ug m 0 an acetone-solid CO, bath. The tissue was used after 2 hours’

760

J. A. V. BUTLER AND D. J. R. LAURENCE

storage. It was partially thawed in 600 ml. ice cold saline (0-9 per cent w/v
pH 3-5) and homogenised in a Waring blender run at full speed for 2 minutes’.
The nucleoprotein was separated by centrifuging for 30 minutes at 800 g., the
supernatant liquid being retained. The nucleoprotein was washed once in
acidific saline and twice in 70 per cent ethanol. The nucleoprotein was then
extracted overnight with 200 ml. 0-25 N—HCl in 70 per cent ethanol in a ball
mill.

The histone contained in the acid extract was clarified (1500 g, 30 minutes)
at room temperature. The insoluble residue was extracted with a further 200 ml.
acid ethanol. The arginine-rich histone was then precipitated from the com-
bined extracts by dialysis against several changes of absolute alcohol, the pre-
cipitate being dried -with acetone.

Arginine-rich histone from mouse Ehrlich ascites {Method B)

Mice had received an injection of Elirlich ascites cells 7 days before injection
with 5 go. amino acid each. The ascites fluid was obtained within a few minutes
after IdUing the animals and the cells were washed twice with 0-9 per cent NaCl
using a centrifuge run at low speed. The washed cells were frozen in liquid
nitrogen and homogenised after partial thawing -with an equal volume of 0-14 m
N aCl containing 0-01 m sodium citrate (pH 7) in a glass homogeniser with a
Teflon plunger. The nuclei were isolated by centrifuging at 1500 g for 10 minutes
and washed twice with the saline citrate mixture. After washing twice with
70 per cent alcohol the nuclei were extracted overnight with 0-25 N — HCl in
70 per cent alcohol by shaking with glass beads. The arginine-rich histone was
precipitated as for rat liver.

Cytoplasmic proteins

After removal of the nuclei or nucleoprotein, a sample of the whole cytoplasmic
protein was precipitated by addition of an equal volume of 10 per cent trichlor-
acetic acid and heating to 90° C. for 10 minutes. The coagulated protein was
washed with 5 per cent trichloracetic acid, alcohol, acetone, ether and then
dried.

RESULTS

Table I shows the specific activities recorded for the various liistones and the
corresponding cytoplasmic fractions prepared in the present work. Values of
H/C are given for each tissue together with the method of preparation.

Table I . — Incorporation of l-Lysine into Histone and Cytoplasmic
Protein of Varwis Tissues*

Tissue

1. Rat liver . . • ■

2. Eat liver . • • •

3. Rat Walker tumour

4. Mouse liver . ■ • •

5. Mouse tumour

G. Ehrlich ascites tumour in mouse
* Values given are

Method of
preparation
of histone
A
B
A
A
A
B

Cj’toplasmjc

protein

(C)

Histone

(H)

H/C

171

120

0-70

115

111

0-95

135

1-46

206

. 112

0-54

129

144

1-12

1270

1220

0-9G

counts per minute/cm= at infinite thickness.

JIET.VBOLIC ACTIVITIES OF HISTONES

761

In Table II are given amino acid analyses for liistonos prepared by the various
methods described, together ivitli some analyses of the cytoplasmic fractions^
The histone preparations contain 23-25 per cent of basic amino acids, and 13-15
per cent acidic amino acids, ■which is within the range tj’piical of histones (7).
The cytoplasmic protein fractions contain 15 per cent of basic amino acids and
about 21 per cent of acidic amino acids.

Table II . — Amino Acid Analyses Given as Moles of Amino Acid
per 100 Residues Determined

Histones

Jloaso

Cytopla.smic proteins

House

Crocker

Rat

A, ^

liver

tumour

liver

Rat

Mouse

Amino acid

(Method A)

(Method B)

liver

tumour

Asp

.5-9

4-8

G-8

8-4

11-0

Glut

7-r>

8-0

8-3

121

10-G

Gly .

9-0

8-7

C-8

8-4

6-9

Ala

120

12-4

13-3

8-7

8-9

Val

7-7

7-i>

5-G

7-9

G-9

Leu

11-1

12-2

12-7

14-9

13-4

. 5-9

5-9

.5-3

G-3

6-5

Thr .

6-3

G-3

5-5

The

1-7

1-9

2-8

4-5

3-8

Tjt

1-7

2-2

2-0

2-4

4-3

Pro

5-2

4'7

G-8

5-G

5-2

His

2-3

2-7

1-8

1-9

Lys .

14-0

13-9

11-4

8-1

8-3

Arg .

8-9

9-2

5-C

5-2

Jlet .

0-9

DISCUSSION

. results are in agreement with the finding of Busch et al. (1958, 1959) in
13' H/C is appreciably greater in the tumours than in normal tissues of the
Is^ving out of consideration the Ehrlich ascites tumour,
1 C IS not comparable in that the amino acid was applied directly -within the
umour, it can be seen that the activities of the cytoplasmic proteins from liver
dTF similar animals, differ more than those of the histones. The

therefore to a considerable extent due to variations of C
hist ' ^3nnot be concluded -with certainty from these results that the tumour
iiormal^^^^ markedly more active metaboheaUy than the histones of comparable

explanation of these results is that the tissue cytoplasm is
norm'll in ifs ability to incorporate amino acids as compared -with the

ascite*^ t same animal. The experiment -with the mouse Ehrlich

iiicorn ^ special case and a direct comparison of the actual amoimts

H /C is°^^ f other mouse tissues is meaningless. The value of

is lunrl^m different, however, from that of the other mouse tumour and

*1 y greater than H/C for mouse liver. This tissue is therefore not in

762

J. A. V. BUTLEK AND D. J. K. LAUEENCE

contradiction to the other cases. The actual levels of labelling in the different
cases (except the ascites tumour) are surprismgly similar.

In conclusion ve maj^ comment on the fact that the degree of labelling of the
histones is in all cases comparable -svith (and in tumours greater than) that of
the cjdoplasmic proteins. A moderate^ liigh degree of metabolic activit}’^ of
histones was observed in early experiments by Daly, AUfrey and Jlirsky (1952),
Brunish and Luck (1952) and Smellie, Mclndoe and Davidson (1953). In these
experiments the degree of labelling of the histone fraction was usually 1 /3 that
of the cjdoplasmic proteins and although the histone was often the least labelled
fraction the activity was higher than could be accounted for as due to the rate
of s3mtbesis required for replication of clrromosomes in cell division.

The activity observed in the cjdoplasmic proteins will depend on the mode
of preparation and will varj”^ with the amount of inactive structural protein
included. Even in the cj^oplasmic particles there exist proteins with very dif-
ferent rates of incorporation (Cohn and Butler, 1958) and it ma}^ well be that the
activities of the c3'toplasmic proteins quoted are well below those of the most
active fractions. Nevertheless, the relatAe incojporation in tumour and liver
found here can hardly be explained except on the assumption that turnover or
s3mthesis of histones occurs besides that necessar3'^ for cell division.

A number of authors (e.g. Stevens, Daoust and Leblond, 1953) have shown
that incorporation into DNA of various normal tissues is proportional to the
mitotic rate. For a 200 g. rat, Stevens ef al. estimated that 0-71 per cent of
liver cells divide per da3q whereas for a rapidl3’' growing tumour almost all the
cells ma3^ divide once in 24 hours and some ma5’’ divide twice. If the incorporation
of histone were proportional to the mitotic rate, a difference of 100-fold would
be expected between the incorporation into the histone of tumour and liver.
The difference found is ver3" much less than this, and from the results of Greenberg
and Sassenrath (1955) the distribution of amino acids after intraperitoneal injec-
tion appears to be too uniform to introduce a large deviation from the expected
difference of this order. Therefore the rate of incorporation into liver histone is
greater than could be expected for the s3mthesis of chromosome histone neces-
sary to maintain the mitotic cycle, and we must conclude that histones undergo
an appreciable amount of turnover or additional S3mthesis.

SUJrMAKY

1. The incorporation of 1-lysine into histones prepared b3' two different
methods and into c3doplasmic proteins of rat and mouse tumoius as well as
rat and mouse liver has been studied under comparable conditions.

2 The ratio of the specific activity of histone (H) to that of the cytoplasmic
proteins (C) of the same ceUs is greater for the tumours than for the liver of the
same animal. However, the differences in these ratios appear to be due to
differences of C to an even greater extent than to differences of H and it is possible
that in tumour cells the rate of incorporation of amino acids into the c3doplasraic

^"^"X'The iSencS rates of incorporation in the tumours and similar normal
tissues do not appear to be related to mitotic rates. It is necessary to conclii •
that a significant turnover or s3mthesis of histone occurs m resting cel .

JIETABOLIC ACIWITIES OE HISTONES

763

We are indebted to ]\Iiss P. Simson for carrying out tire amino acid analyses
given ; to Messrs. B. Mitclilej’- and 0. Smitli for implanting the tumours.

This investigation has been supported b 3 '^ grants to the Chester Beatty Research
Institute (Listitute of Cancer Research : Royal Cancer Hospital) from the
Medical Research Council, the British Empire Cancer Campaign, the Jane Coffin
Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the
National Cancer Institute of the National Institutes of Health, U.S. Public
Health Service.

REFERENCES

Beumsh, R. and Luck, J. M. — (1952) J. biol. Chem. 198, 621.

Busch, H., Davis, J. R. and Anderson, D. C. — (1958) Cancer Res., 18, 91P
Idem, ^)A^^s, J. R., Honig, G. R., Anderson, D. C., Nair, P. V. and isiviiAN, W. L.
—(1959) Ibid., 19, 1030.

Cohn, P. and Butler, J. A. V. — (1958) Biochem. J., 70, 254.

Daly, M. M., Allfkey, V. G. and Mtrsky', A. E. — (1952) J. gen. Physiol., 36. 173.
DA^^DSON, P. F.— (1957) Biochem. J., 66, 708.

Greenberg, D. M. and Sassenrath, E. N. — (1955) Cancer Res., 15, 620.

Johns, E. MC, Phillips, D. M. P., Sehson, P. and Butler, J. A. V. — (1960) Biochem.
J., 77, 631.

LoFTnELD, R. B. AND HARRIS, A.— (1956) J. biol. Chem., 219, 151.

Phillips, D. M. P. and Johns, E. W.— (1959) Biochem. J., 72, 538.

Rotherham, J., Irvin, J. C., Ibitn, E. M. and Holbrook, D. J. — (1957) Proc. Soc.
exp.Biol.,N.Y.,^%,2\.

Smellie, R. M. S., McIndoe, V. M. and Daihdson, J. N. — (1953) Biochim. biophys.
Acta, 11, 559.

Stevens, C. E., Daoust, R. and Leblond, C. P.— (1953) J. biol. Chem., 202, 177.

INDEX TO AUTHORS

AcEraDO, A. R., see Holland, 11. H.

Acs, Th., see Csaba, G.

Allisok, a. C. and Armstrong, J. A., nb-
normal distribution of nucleic acid in
tissue culture cells infected with polyoma
\Trus, 313

Antoni, F., see Hidvegi, E. .J.

Armstrong, J. A., see Allison, A. C.

Baanders-van Halewun, E. A., see de
Viaard, F.

Baldmtn, E. W’., see Bitenshy, L.

Bail, J. K. and McCarter, J. A., a study
of dose and effect in initiation of skin
tumours by a carcinogenic liydro-
carbon, 577

Barker, J. F., see Ghadialhj, F. N.

Baserga, R., Putong, P. B., Tyler, H. and
Wartman, W. B., the dose-response
relationship between the number of
embolic tumor cells and the incidence of
blood-borne metastases, 173

Belcher, E, H. and Sijipson, S. M., radio-
active tracer studies of red cell destruc-
tion in rats bearing a transplantable
tumour, 224

Bhavsar, JI. B., see Sahasrabudhe, M. B.

riiANcinoRi, C. and Caschera, F., the
gradual conversion of a spontaneous
mouse mammaiy carcinoma to an
ascites tumour, 668

ielschowsky", F. and Bielschowsky, M.,
carcinogenesis in the pituitary dwarf
mouse. The respon.se to 2-amino-
nuorene, 195

ielschowsky, M., see Bielschowsky, F.
Bitensky, L., Baldwtn, R. W. and
lhaa'en, .j., a histochemical study of
le early stages of carcinogenesis in rat
liver. Localization of fluorescent car-
cinogen and changes in succinic de-
_ iiydrogenase activity, 690

TT , a histochemical study

o no early stages of carcinogenesis in
ra liver. Changes in cellular lipiiLs
and mitochondria, 696
Bi-air, D. W., see Sim, A. IF.

''onmxcTON, M. M., ComiELL, R. H. and
‘ .’’Hi'ic.s, A. I., development of car-
cinoma of the cervix uteri. Obser\-ations
B'om cytological examination
'I 10,000 cervical smears, 1.51

Boyi.and. E. see Grrenimj IF. P.

Brew, D. St..1. nnd .Iack.son, .1. G.,

lyiniiho.snrcoma in (he ovary in young
Afrienn girls in Nigeria, 021
Bin-LER, J. A. V. and Laurence, D. J. R.
relative metabolic activities of histones
in tumours and liver, 758

Caecutt, G., Do.vey, D. nnd Coates, .7.,
the effects of some chemical carcinogens
upon the — SH levels of target nnd
non-target tissues, 746

Carr, J. G., kidney carcinomas of the fowl
induced by the MH2 reticulocndo-
thclioma virus, 77

Ca-schera, F., sec Bianrifiori, C.

CiLASEN, W. H., sec Lemon, H. M.

Chay’en, j., sec Bilcnsky, L.

Cherry, C. P. nnd Glucksmann, A„ the
effect of endocrine changes of irradia-
tion and of additional treatment of the
skin on the induction of tumours in the
female genital tract of rats by chemical
carcinogens, 489

Chesterman, F. C., see Negroni, G.

Clark, C. M. and Goodlad, G. A. J., the
influence of diet on the action of the
Walker 256 carcinoma on liver protein
and nucleic acids, 327

Clark, D. A., see Holland, B. H.

Clemo, G. R. and Miller, E. W., tumour
promotion by the neutral fraction of
cigarette smoke, 651

Coates, J., see Calcuft, G.

Coutjell, R. H., see Boddington, M. M.

Csaba, G., Acs, Th., Horvath, C. and
Kara, E., some new data concerning
the biology of tumours. The effects of
heparin inhibitors on tumour growth,
367

, Horvath, C. and Acs, Th., some

new data concerning the biology of
tumours. The effects of heparin and its
components on tumour grou-th, 362

Dale, R. A., the Isolation of epithelial cells
from normal and neoplastic colon, 45
Darcy, D. A., a quantitative study of a
serum protein associated with tissue
growth. Levels found in rats under
various phy.siological conditions, 524

766

INDEX

Dakcy, D. a., a quantitative stud 3 '’ of a
serum protein associated with tissue
growth. Vaiiies found in tumour-bearing
rats, 534

Davies, R. I., see Stocks, P.

Davies, W. and Wilmshuhst, J. R., car-
cinogens formed in t)ie heating of food-
stuffs. Formation of 3,4-benzopjTene
from stareij at 370-390° C., 295

Do.vev, D., see Calmtt, 6.

Dbayton, H. a., the effect of incubation on
tliree fowl tumour viruses, 306

Ellis, I. R., see Israe/, J/.S.

Englander, O, and Sarangi, A., effect of
Thio-TEPA on advanced malignant
ovarian tumours, 28

Ferrari, E. and Kreyberg, L., liistological
tj^ies in a lung cancer material in
Venice. 609

Forrest, A. P. M., see Sim, A. II'.

Fra.ser, L. E., see Young, S.

(Jhadially, F. X., carcinogeno.si.s in the .skin
of the hedgehog, 212

, see lllman, 0.

, Illman, O. and Barker, J. F., the

effect of trauma on the melanotic
tumours of the hamster, 647

Gillman, j., see Mirvish, S.

Gluoksmann, a., see Cherry, C. P.

Godsmabk, B., see Greening, IF. P.

Goodlad, G. a. j., see O/ark, C. M.

GraIG, F. a., see Siegel, E.

Greening, IV. P., Ra.msay, G. S.,
Stevenson, J. J., Boyland, E.,
Rigby-Jones, P. C. and Godsmabk,
B., results in the treatment of cancer of
the breast by interstitial irradiation of
the pituitary, 627

Gkeiseach, W. E. and Purves, H. D.,
basophil adenomata in the rat bj'po-
phj^sis after gonadectomjL 49

Hakim, S. A., see Ranadive, K. J.

Hanbury', W. j., seoondarj^ tumours of the
heart, 23

Heath, J. C., tlie histogenesis of malignant
tiunoiu'S induced bj' cobalt in the rat,
478

Herdan, G., the frequency of cancer in
diabetes mellitus, 449

Hewitt, H. B. and Wilson, C. W., furtlier
studies relating to the implications of
radiation survival curve data for treat-
ment of CBA mouse leukaemia by
whole-bod 3 m'rradiation, 186

HiDvkGi, E. J., Antoni, F. and Lapis, K.,
effect of cheinotherapeutics on the
nucleic acid metabolism of tumours.
Incorporation of into nucleic acids
following treatment with degranoi, 139
Hill, K. R., see Hirchinson, F.
Hihchinson, V. and Hill, K. R., the effects
of a senecio alkaloid (monocrotaline) on
human embryo liver in tissue culture.
637

Hobkirk, R., see Sim, A. IF.

Holland, R. H., Acevedo, A. R. and
Clark, D. A., the arsenic content of
bronchial mucosa and submuco.sa in
man. A comparison of specimens from
lung cancer victims and control tissue,
169

Horvath, C., see Gsaha, 6.

Houghton, P. and JIabtin, X, H., the
m 3 ’eloma globulins, 709
Howell, .1. S., the chemical induction of
breast tumours in the rat : hormonal
factors in tumour production, 657
Huh, T. Y. and McCarter, J. A., phenan-
threne a.s an anti-initiating agent, .591

Illman, 0., see Ohadialhj, F. R.

and Ghadially', F. X., coat oolow

and experimental melanotic tumoiu-
production in the hamster, 483
Israel, M. S. and Ellis, I. R., the neo-
plastic potentialities of mouse thyroid
under extreme stimulation, 206
Ivebsen, S., see Pullinger, B. D.

Jackson. J. G., see Brew, D. St.J.

Kapa, E., see Gsaba, G.

Kharkab, K. R., see Ranadive, K. J.
Kelsian, M.. see Lemon, H. M.

Kjrby', K. S., induction of tumours 65 '
tannin extracts, 147
Kirk, R., see Muir, C. S.

Krey'bbrg, L., see Ferrari, E.

BE Laivb, j. M'. j., see de Waard, F.

Lapis, K., see Hidvegi, E. J.

L.aurence, D. j. R., see Butler, J. A- \ ■
Lemon, H. M., Mueller, J. H., Looney’,
J. M., Ch-ysen, W. H. and ILelman, M-,
h 3 q)ercitricemia inhuman cancer. Factors
concerned in patliogenesis and treatment.
376

Lews, F. J. W. and Plaice, C. H- J-
urinar 3 ’ jS-g!ucuronidase actii’itY' ”>
cancer of the bladder and other diseases,
106

Looney', J. M., sec Lemon, H. M-
Louis, C. J., fluorescein-globulin affinities ot
the Shope papilloma, 216

INDEX

767

Lucas, R. B., see Thackrmj, A. C.

Lyons, M. J. and Spence, J. B., environ-
mental free radieals, 703

McCabtbe, j. a., see BaU, J ■ K.

■ — ^ — ■, see Huh, T. Y.

ilARCHANT, J., the development of ovarian
tumours in ovaries grafted from mice
pretreated with dimetli 5 ’lbenzanthra-
cene. Inliibition by the presence of
norma! ovarian tissue, 514

^ the development of ovarian tiiinoui-s

in ovaries grafted from mice pretreated
with diemethylbenzanthracene. The
effects of the grafting operation itself
and the effects of gi-afting ovaries from
mice at early stages in pretreatment
rrith the carcinogen, 519
JIartin, N. H., see Houghton, P.

JIaetins, a. G., adenocarcinoma of the
uterus in infancj% 165
JIiKULSKA, Z. B., see O'Gorman, P.
liliLLEE, E. W., see Clemo, G. R.

Miller, J. F. A. P., studies on mouse leu-
kaemia. Leukaemogenesis by cell-free
filtrates inoculated in newborn and adult
mice, 83

, studies on mouse leukaemia. The

role of the thymus in leukaemogenesis
by cell-free leukaemio filtrates, 93

, studies on mouse leukaemia. The

fate of thymas homografts in immuno-
logically tolerant mice, 244
ilmvisH, S. and Gillman, J., bile acid
composition and bile volume in butter
yellow fed rats in relation to liver
cancer, 346

Mody, .T. K., the action of four carcinogemc
hydrocarbons on the ovaries of IF mice
and the histogenesis of induced tumours,
256

Montemuebo, D. G., the effect of nitro-
furazone on the testes and accessory sex
organs of normal rats and rats bearing
the Walker carcinoma 256, 319
Mueller, J. H., see Lemon, H. M.

Muir, C. S., cancer of the lung, trachea and
larynx in Singapore, I

~ ■ ’’■nd Kirk, R., betel, tobacco, and

cancer of the mouth. 597

NarurKvUi, M. V., .see Sahasrabudhe, M. B.
^EGRONI, G. and Chesterman, F. C.,
virus-cell relationship in kidney
tumours induced in golden hamsters by
the Mill Hill polyoma virus, 672
. Eisii, W. J. P. and Rylett, A., accumula-
tion of phosphoethanolamine in the
livcre of rats injected with hepato-
••arcinogens, 737

Nehukkar, M. K., sec Sahasrabudhe, M . B.

O’Gorman, P., the in vitro cytotoxic activity
of murine iso-antisera on normal and
malignant colls, 335

_ _ _ and JIiKUL-sKA, Z. B., the antigenic
structure of a transplanted sarcoma, 121

Patey, D. H., early (prophylactic) oophor-
ectomy and ndrpnalcctom 3 ’’ in car-
cinoma of the breast ; an interim
report, 457

Pearson, A. E. G., serial irradiation of
mouse tumours : some changes in
histologj’ and cj'tolog,v, 200

Plaice, C. H. J., see Lewis, F. .7. 11’.

Porter, K. A., graft-vei-sus-host reactions
in the rabbit, 66

Powell, A. K., studies on fluid media for
the cultivation of mouse aseite.s tumour
cells in vitro, 99

Pre.snov, M. a., histological and histo-
chemicat changes induced b>' alkj'lating
agents in transplanted rat sarcoma with
special reference to sarcolj'sine, 60

PuLLiNGER, B. D., tests foi' mammaiy
tumour agent in and RIII/ mouse

strains, 279

, reduction of mammarj’ carcinoma

and adenoma in breeders after

late ovariectomj’, 502

and IvERSEN, S., mammary tumour

incidence in relation to age and number
of littere in CjH/ and RIII/ mice, 267

PuBVES, H. D., see Griesbach, W. E.

PuTONG, P. B., see Baserga, R.

Ramsay, G. «S., see Greening, IT. P.

Ranadive, K. j., Hakim, S. A. and
Kharkae, K. R., chemical induction
of mammaiy cancer in pseudopreg-
nancy, 508

Revesz, L,., see Seelig, K. H.

Rigby-Jones, P. C., see Greening, IF. P.

Roberts, D. C., see Trevan, D. J.

and Trevan, D. . 1 ., cell division in

an ascites tumour in vitro with especial
reference to abnormalities of ej'to-
plasmic cleavage, 716

Rylett, A., see Neish, W. J. P.

Sachs, B. A., see Siegel, E.

Sahasrabudhe, M. B., Nerubkar, M. K.,
Narurkar, M. V., Tilak, B. D. and
Bhavsar, M. D., inliibition of tumoui’
growth by interference of hexose-mono-
phosphate pathway. Synthesis and
anticancer properties of thiophene 2 : 5
dicarboxjdic acid, 547
Sarangi, a., see Englander, O.

Si;i'.i,i(!. K. .1. and Hjivksz, L., ofTcct of
li'llially tlainanod tiiinoiir oolls tipon the
j^rowlh of adniixf'd viajjlc ooIIh in
tliffnsinn oliainboi-s, 120

Sir.ciin., Sachs, H. A. and Guak:, F. A.,
fate of ^^S-(:vstin<‘ in imtltij)lo nivcloma,
700

SiM. A. ^V., Hoiikiiik, ]{., Sti:\vaiit, II. .1..
Bi.aiu, D. W. and Foiiar.sT, A. P. M.,
urinary IT-kotostoroids and (lioir frac-
tions ii\ women with breast eaneer
tn-ated by endoerine surgery, 400

SlMi’.so.v, ,S. HI., see Hclrficr, E. I/.

Si’KNCi;, .7. H., see Lyimt), M . ,/.

SniKJOS, A. 1., see liw{rlliiiit(»i, .7/. M.

Staszkwski, .1., sinokiiif; and eaneer in
Poland, 419

S'n:tN'-7Vi;itrir.o\vsKA', K., indtiefion of eaneer
of the cervix uteri in relation to the
oestnis cycle, 000

Stkwaut, II. .7., see Siiii, A. fl'.

Stkvknsox, .7. .7., see Clrceniiuj, II'. J‘.

Stocks, P., on the relations between atino-
spberic pollution in urban and rural
localities and mortality from cancer,
bronchitis and pneumonia, with ])ar-
tictdar reference to 0:4 benzopyrene,
beryllium, molybdenum, vanadium and
arsenic, 097

■■ • and Daviks, It. I., epidemiological
evidence from ebemical and spcctro-
graphic analyses that soil is concerned
in the causation of cancer, 8

Stoker, HI., studies on the oncogenic
activity of the Toronto strain of
polj’oina virus, 679

. 'ITrACKiiAy, A. C. and Lucas, K. B., the
histology of cylindroma of mucous gland
origin, 012

T'iiomlixsox, R. H., an experimental
method for comparing treatments of
intact malignant tumours in animals
and its application to the use of oxj'gen
in radiotherapy, 555
'JTi.ak, 75. D., see Sahaarabudhe, M. B.
'ruEVAN, D. >7., SCO Roberts, D. C.

and Roberts, D. C., sheet fohnation

Ijy cells of an ascites tumour in vitro, 724
Ta’I.ku, S., see Baserga, R.

DE HVaard, F., de Laiat:, J. W. J. and
Raaxders-van Haieaatjn, E. A., on
the bimodal age distribution of mam-
mary carcinoma, 437
Wautman, HV. B., see Baserga, R.
Whitaker, B. L., plasma ^-glucuromdase
levels in breast cancer, 471
WiiiM.SHURST, J. R., see Davies, 71^
WiLSox, C. W; see Hewitt, H. B.

Youxo, S. and Fraser, L. E., e-xp^encM
with h>’pophysectomy in mice. Criteria
of complete removal, 285.

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